Oligonucleotide analogues

ABSTRACT

Novel oligomers, and synthesis thereof, comprising one or more bi-, tri, or polycyclic nucleoside analogues are disclosed herein. The nucleoside analogues have a “locked” structure, termed Locked Nucleoside Analogues (LNA). LNA&#39;s exhibit highly desirable and useful properties. LNA&#39;s are capable of forming nucleobase specific duplexes and triplexes with single and double stranded nucleic acids. These complexes exhibit higher thermostability than the corresponding complexes formed with normal nucleic acids. The properties of LNA&#39;s allow for a wide range of uses such as diagnostic agents and therapeutic agents in a mammal suffering from or susceptible to, various diseases.

FIELD OF THE INVENTION

[0001] The present invention relates to the field of bi- and tricyclicnucleoside analogues and to the synthesis of such nucleoside analogueswhich are useful in the formation of synthetic oligonucleotides capableof forming nucleobase specific duplexes and triplexes with singlestranded and double stranded nucleic acids. These complexes exhibithigher thermostability than the corresponding complexes formed withnormal nucleic acids. The invention also relates to the field of bi- andtricyclic nucleoside analogues and the synthesis of such nucleosideswhich may be used as therapeutic drugs and which may be incorporated inoligonucleotides by template dependent nucleic acid polymerases.

BACKGROUND OF THE INVENTION

[0002] Synthetic oligonucleotides are widely used compounds in disparatefields such as molecular biology and DNA-based diagnostics andtherapeutics.

[0003] Therapeutics

[0004] In therapeutics, e.g., oligonucleotides have been usedsuccessfully to block translation in vivo of specific mRNAs therebypreventing the synthesis of proteins which are undesired or harmful tothe cell/organism. This concept of oligonucleotide mediated blocking oftranslation is known as the “antisense” approach. Mechanistically, thehybridising oligonucleotide is thought to elicit its effect by eithercreating a physical block to the translation process or by recruitingcellular enzymes that specifically degrades the mRNA part of the duplex(RNAseH).

[0005] More recently, oligoribonucleotides and oligodeoxyribonucleotidesand analogues thereof which combine RNAse catalytic activity with theability to sequence specifically interact with a complementary RNAtarget (ribozymes) have attracted much interest as antisense probes.Thus far ribozymes have been reported to be effective in cell culturesagainst both viral targets and oncogenes.

[0006] To completely prevent the synthesis of a given protein by theantisense approach it is necessary to block/destroy all mRNAs thatencode that particular protein and in many cases the number of thesemRNA are fairly large. Typically, the mRNAs that encode a particularprotein are transcribed from a single or a few genes. Hence, bytargeting the gene (“antigene” approach) rather than its mRNA productsit should be possible to either block production of its cognate proteinmore efficiently or to achieve a significant reduction in the amount ofoligonucleotides necessary to elicit the desired effect. To blocktranscription, the oligonucleotide must be able to hybridise sequencespecifically to double stranded DNA. In 1953 Watson and Crick showedthat deoxyribo nucleic acid (DNA) is composed of two strands (Nature,1953, 171, 737) which are held together in a helical configuration byhydrogen bonds formed between opposing complementary nucleobases in thetwo strands. The four nucleobases, commonly found in DNA are guanine(G), adenine (A), thymine (T) and cytosine (C) of which the G nucleobasepairs with C, and the A nucleobase pairs with T. In RNA the nucleobasethymine is replaced by the nucleobase uracil (U) which similarly to theT nucleobase pairs with A. The chemical groups in the nucleobases thatparticipate in standard duplex formation constitute the Watson-Crickface. In 1959, Hoogsteen showed that the purine nucleobases (G and A) inaddition to their Watson-Crick face have a Hoogsteen face that can berecognised from the outside of a duplex, and used to bind pyrimidineoligonucleotides via hydrogen bonding, thereby forming a triple helixstructure. Although making the “antigene” approach conceptually feasiblethe practical usefulness of triple helix forming oligomers is currentlylimited by several factors including the requirement for homopurinesequence motifs in the target gene and a need for unphysiologically highionic strength and low pH to stabilise the complex.

[0007] The use of oligonucleotides known as aptamers are also beingactively investigated. This promising new class of therapeuticoligonucleotides are selected in vitro to specifically bind to a giventarget with high affinity, such as for example ligand receptors. Theirbinding characteristics are likely a reflection of the ability ofoligonucleotides to form three dimensional structures held together byintramolecular nucleobase pairing.

[0008] Likewise, nucleosides and nucleoside analogues have proveneffective in chemotherapy of numerous viral infections and cancers.

[0009] Also, various types of double-stranded RNAs have been shown toeffectively inhibit the growth of several types of cancers.

[0010] Diagnostics

[0011] In molecular biology, oligonucleotides are routinely used for avariety of purposes such as for example (i) as hybridisation probes inthe capture, identification and quantification of target nucleic acids(ii) as affinity probes in the purification of target nucleic acids(iii) as primers in sequencing reactions and target amplificationprocesses such as the polymerase chain reaction (PCR) (iv) to clone andmutate nucleic acids and (vi) as building blocks in the assembly ofmacromolecular structures.

[0012] Diagnostics utilises many of the oligonucleotide based techniquesmentioned above in particular those that lend themselves to easyautomation and facilitate reproducible results with high sensitivity.The objective in this field is to use oligonucleotide based techniquesas a means to, for example (i) tests humans, animals and food for thepresence of pathogenic micro-organisms (ii) to test for geneticpredisposition to a disease (iii) to identify inherited and acquiredgenetic disorders, (iv) to link biological deposits to suspects in crimetrials and (v) to validate the presence of micro-organisms involved inthe production of foods and beverages.

[0013] General Considerations

[0014] To be useful in the extensive range of different applicationsoutlined above, oligonucleotides have to satisfy a large number ofdifferent requirements. In antisense therapeutics, for instance, auseful oligonucleotide must be able to penetrate the cell membrane, havegood resistance to extra- and intracellular nucleases and preferablyhave the ability to recruit endogenous enzymes like RNAseH. In DNA-baseddiagnostics and molecular biology other properties are important suchas, e.g., the ability of oligonucleotides to act as efficient substratesfor a wide range of different enzymes evolved to act on natural nucleicacids, such as e.g. polymerases, kinases, ligases and phosphatases. Thefundamental property of oligonucleotides, however, which underlies alluses is their ability to recognise and hybridise sequence specificallyto complementary single stranded nucleic acids employing eitherWatson-Crick hydrogen bonding (A-T and G-C) or other hydrogen bondingschemes such as the Hoogsteen mode. The are two important terms affinityand specificity are commonly used to characterise the hybridisationproperties of a given oligonucleotide. Affinity is a measure of thebinding strength of the oligonucleotide to its complementary targetsequence (expressed as the thermostability (T_(m)) of the duplex). Eachnucleobase pair in the duplex adds to the thermostability and thusaffinity increases with increasing size (No. of nucleobases) of theoligonucleotide. Specificity is a measure of the ability of theoligonucleotide to discriminate between a fully complementary and amismatched target sequence. In other words, specificity is a measure ofthe loss of affinity associated with mismatched nucleobase pairs in thetarget. At constant oligonucleotide size the specificity increases withincreasing number of mismatches between the oligonucleotide and itstargets (i.e. the percentage of mismatches increases). Conversely,specificity decreases when the size of the oligonucleotide is increasedat a constant number of mismatches (i.e. the percentage of mismatchesdecreases). Stated another way, an increase in the affinity of anoligonucleotide occurs at the expense of specificity and vice-versa.

[0015] This property of oligonucleotides creates a number of problemsfor their practical use. In lengthy diagnostic procedures, for instance,the oligonucleotide needs to have both high affinity to secure adequatesensitivity of the test and high specificity to avoid false positiveresults. Likewise, an oligonucleotide used as antisense probes needs tohave both high affinity for its target mRNA to efficiently impair itstranslation and high specificity to avoid the unintentional blocking ofthe expression of other proteins. With enzymatic reactions, like, e.g.,PCR amplification, the affinity of the oligonucleotide primer must behigh enough for the primer/target duplex to be stable in the temperaturerange where the enzymes exhibits activity, and specificity needs to behigh enough to ensure that only the correct target sequence isamplified.

[0016] Given the shortcomings of natural oligonucleotides, newapproaches for enhancing specificity and affinity would be highly usefulfor DNA-based therapeutics, diagnostics and for molecular biologytechniques in general.

[0017] Conformationally Restricted Nucleosides

[0018] It is known that oligonucleotides undergo a conformationaltransition in the course of hybridising to a target sequence, from therelatively random coil structure of the single stranded state to theordered structure of the duplex state.

[0019] A number of conformationally restricted oligonucleotidesincluding bicyclic and tricyclic nucleoside analogues (FIGS. 1A and 1Bin which B=nucleobase) have been synthesised, incorporated intooligonucleotide and oligonucleotide analogues and tested for theirhybridisation and other properties. Bicyclo[3.3.0] nucleosides (bcDNA)with an additional C-3′,C-5′-ethano-bridge (A and B) have beensynthesised with all five nucleobases (G, A, T, C and U) whereas (C) hasbeen synthesised only with T and A nucleobases (M. Tarköy, M. Bolli, B.Schweizer and C. Leumann, Helv. Chim. Acta, 1993, 76, 481; Tarköy and C.Leumann, Angew. Chem., Int. Ed. Engl., 1993, 32, 1432; M. Egli, P.Lubini, M. Dobler and C. Leumann, J. Am. Chem. Soc., 1993, 115, 5855; M.Tarköy, M. Bolli and C. Leumann, Helv. Chim. Acta, 1994, 77,716; M.Bolli and C. Leumann, Angew. Chem., Int. Ed. Engl., 1995, 34, 694; M.Bolli, P. Lubini and C. Leumann, Helv. Chim. Acta, 1995, 78, 2077; J. C.Litten, C. Epple and C. Leumann, Bioorg. Med. Chem. Lett, 1995, 5, 1231;J. C. Litten and C. Leumann, Helv. Chim. Acta, 1996, 79, 1129; M. Bolli,J. C. Litten, R. Schültz and C. Leumann, Chem. Biol., 1996, 3, 197; M.Bolli, H. U. Trafelet and C. Leumann, Nucleic Acids Res., 1996, 24,4660). DNA oligonucleotides containing a few, or being entirelycomposed, of these analogues are in most cases able to form Watson-Crickbonded duplexes with complementary DNA and RNA oligonucleotides. Thethermostability of the resulting duplexes, however, is either distinctlylower (C), moderately lower (A) or comparable to (B) the stability ofthe natural DNA and RNA counterparts. All bcDNA oligomers exhibited apronounced increase in sensitivity to the ionic strength of thehybridisation media compared to the natural counterparts. Theα-bicyclo-DNA (B) is more stable towards the 3′-exonuclease snake venomphosphordiesterase than the β-bicyclo-DNA (A) which is only moderatelymore stable than unmodified oligonucleotides.

[0020] Bicarbocyclo[3.1.0]nucleosides with an additional C-1′,C-6′- orC-6′,C-4′-methano-bridge on a cyclopentane ring (D and E, respectively)have been synthesised with all five nucleobases (T, A, G, C and U). Onlythe T-analogues, however, have been incorporated into oligomers.Incorporation of one or ten monomers D in a mixed polypyrimidine DNAoligonucleotide resulted in a substantial decrease in the affinitytowards both DNA and RNA oligonucleotides compared to the unmodifiedreference oligonucleotide. The decrease was more pronounced with ssDNAthan with ssRNA. Incorporation of one monomer E in two differentpoly-pyrimidine DNA oligonucleotides induced modest increases in T_(m)'sof 0.8° C. and 2.1° C. for duplexes towards ssRNA compared withunmodified reference duplexes. When ten T-analogues were incorporatedinto a 15 mer oligonucleotide containing exclusively phosphorothioateinternucleoside linkages, the T_(m) against the complementary RNAoligonucleotide was increased approximately 1.3° C. per modificationcompared to the same unmodified phosphorothioate sequence. Contrary tothe control sequence the oligonucleotide containing the bicyclicnucleoside E failed to mediate RNAseH cleavage. The hybridisationproperties of oligonucleotides containing the G, A, C and U-analogues ofE have not been reported. Also, the chemistry of this analogue does notlend itself to further intensive investigations on completely modifiedoligonucleotides (K. -H. Altmann, R. Kesselring, E. Francotte and G.Rihs, Tetrahedron Lett., 1994, 35, 2331; K. -H. Altmann, R.Imwinkelried, R. Kesselring and G. Rihs, Tetrahedron Lett., 1994, 35,7625; V. E. Marquez, M. A. Siddiqui, A. Ezzitouni, P. Russ, J. Wang, R.W. Wagner and M. D. Matteucci, J. Med. Chem., 1996, 39, 3739; A.Ezzitouni and V. E. Marquez, J. Chem. Soc., Perkin Trans. 1, 1997,1073).

[0021] A bicyclo[3.3.0] nucleoside containing an additionalC-2′,C-3′-dioxalane ring has been synthesised as a dimer with anunmodified nucleoside where the additional ring is part of theinternucleoside linkage replacing a natural phosphordiester linkage (F).This analogue was only synthesised as either thymine-thymine orthymine-5-methylcytosine blocks. A 15-mer polypyrimidine sequencecontaining seven of these dimeric blocks and having alternatingphosphordiester- and riboacetal-linkages, exhibited a substantiallydecreased T_(m) against complementary ssRNA compared to a controlsequence with exclusively natural phosphordiester internucleosidelinkages (R. J. Jones, S. Swaminathan, J. F. Millagan, S. Wadwani, B. S.Froehler and M. Matteucci, J. Am. Chem. Soc., 1993, 115, 9816).

[0022] The two dimers (G and H) with additional C-2′,C-3′-dioxane ringsforming bicyclic[4.3.0]-systems in acetal-type internucleoside linkageshave been synthesised as T-T dimers and incorporated once in the middleof 12 mer polypyrimidine oligonucleotides. Oligonucleotides containingeither G or H both formed significantly less stable duplexes withcomplementary ssRNA and ssDNA compared with the unmodified controloligonucleotide (J. Wang and M. D. Matteucci, Bioorg. Med. Chem. Lett.,1997, 7, 229).

[0023] Dimers containing a bicyclo[3.1.0]nucleoside with aC-2′,C-3′-methano bridge as part of amide- and sulfonamide-type (I andJ) internucleoside linkages have been synthesised and incorporated intooligonucleotides. Oligonucleotides containing one ore more of theseanalogues showed a significant reduction in T_(m) compared to unmodifiednatural oligonucleotide references (C. G. Yannopoulus, W. O. Zhou, P.Nower, D. Peoch, Y. S. Sanghvi and G. Just, Synlett, 1997, 378).

[0024] A trimer with formacetal internucleoside linkages and abicyclo[3.3.0]glucose-derived nucleoside analogue in the middle (K) hasbeen synthesised and connected to the 3′-end of an oligonucleotide. TheT_(m) against complementary ssRNA was decreased by 4° C., compared to acontrol sequence, and by 1.5° C. compared to a sequence containing two2′,5′-formacetal linkages in the 3′-end (C. G. Yannopoulus, W. Q. Zhou,P. Nower, D. Peoch, Y. S. Sanghvi and G. Just, Synlett, 1997, 378).

[0025] Very recently oligomers composed of tricyclicnucleoside-analogues (L) have been reported to show increased duplexstability compared to natural DNA (R. Steffens and C. Leumann (PosterSB-B4), Chimia, 1997, 51, 436).

[0026] An attempt to make the bicyclic uridine nucleoside analogue Qplanned to contain an additional O-2′,C-4′-five-membered ring, startingfrom 4′-C-hydroxymethyl nucleoside P, failed (K. D. Nielsen,Specialerapport (Odense University, Denmark), 1995).

[0027] Until now the pursuit of conformationally restricted nucleosidesuseful in the formation of synthetic oligonucleotides with significantlyimproved hybridisation characteristics has met with little success. Inthe majority of cases, oligonucleotides containing these analogues formless stable duplexes with complementary nucleic acids compared to theunmodified oligonucleotides. In other cases, where moderate improvementin duplex stability is observed, this relates only to either a DNA or anRNA target, or it relates to fully but not partly modifiedoligonucleotides or vice versa. An appraisal of most of the reportedanalogues are further complicated by the lack of data on analogues withG, A and C nucleobases and lack of data indicating the specificity andmode of hybridisation. In many cases, synthesis of the reported monomeranalogues is very complex while in other cases the synthesis of fullymodified oligonucleotides is incompatible with the widely usedphosphoramidite chemistry standard.

SUMMARY OF THE INVENTION

[0028] In view of the shortcomings of the previously known nucleosideanalogues, the present inventors have now provided novel nucleosideanalogues (LNAs) and oligonucleotides have included LNA nucleosideanalogues therein. The novel LNA nucleoside analogues have been providedwith all commonly used nucleobases thereby providing a full set ofnucleoside analogues for incorporation in oligonucleotides. As will beapparent from the following, the LNA nucleoside analogues and the LNAmodified oligonucleotide provides a wide range of improvements foroligonucleotides used in the fields of diagnostics and therapy.Furthermore, the LNA nucleoside analogues and the LNA modifiedoligonucleotide also provides completely new perspectives in nucleosideand oligonucleotide based diagnostics and therapy.

[0029] Thus, the present invention relates to oligomers comprising atleast one nucleoside analogue (hereinafter termed “LNA”) of the generalformula I

[0030] wherein X is selected from —O—, —S—, —N(R^(N*))—, —C(R⁶R^(6*))—,—O—C(R⁷R^(7*))—, —C(R⁶R^(6*))—O—, —S—C(R⁷R^(7*))—, —C(R⁶R^(6*))—S—,N(R^(N*))—C(R⁷R^(7*))—, —C(R⁶R^(6*))—N(R^(N*)), and—C(R⁶R^(6*))—C(R⁷R^(7*))—;

[0031] B is selected from hydrogen, hydroxy, optionally substitutedC₁₋₄-alkoxy, optionally substituted C₁₋₄-alkyl, optionally substitutedC₁₋₄-acyloxy, nucleobases, DNA intercalators, photochemically activegroups, thermochemically active groups, chelating groups, reportergroups, and ligands;

[0032] P designates the radical position for an internucleoside linkageto a succeeding monomer, or a 5′-terminal group, such internucleosidelinkage or 5′-terminal group optionally including the substituent R⁵;

[0033] one of the substituents R², R^(2*), R³, and R^(3*) is a group P*which designates an internucleoside linkage to a preceding monomer, or a3′-terminal group;

[0034] one or two pairs of non-geminal substituents selected from thepresent substituents of R^(1*), R^(4*), R⁵, R^(5*), R⁶, R^(6*), R⁷,R^(7*), R^(N*), and the ones of R², R^(2*), R³, and R^(3*) notdesignating P* each designates a biradical consisting of 1-8groups/atoms selected from —C(R^(a)R^(b))—, —C(R^(a))═C(R^(a))—,—C(R^(a))═N—, —O—, —Si(R^(a))₂—, —S—, —SO₂—, —N(R^(a))—, and >C=Z,

[0035] wherein Z is selected from —O—, —S—, and —N(R^(a))—, and R^(a)and R^(b) each is independently selected from hydrogen, optionallysubstituted C₁₋₁₂-alkyl, optionally substituted C₂₋₁₂-alkenyl,optionally substituted C₂₋₁₂-alkynyl, hydroxy, C₁₋₁₂-alkoxy,C₂₋₁₂-alkenyloxy, carboxy, C₁₋₁₂-alkoxycarbonyl, C₁₋₁₂-alkylcarbonyl,formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,heteroaryloxycarbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di(C₁₋₆-alkyl)amino, carbamoyl, mono- anddi(C₁₋₆-alkyl)-amino-carbonyl, amino-C₁₋₆-alkyl-aminocarbonyl, mono- anddi(C₁₋₆-alkyl)amino-C₁₋₅-alkyl-aminocarbonyl, C₁₋₆-alkyl-carbonylamino,carbamido, C₁₋₆-alkanoyloxy, sulphono, C₁₋₆-alkylsulphonyloxy, nitro,azido, sulphanyl, C₁₋₆-alkylthio, halogen, DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands, where aryl and heteroaryl may beoptionally substituted, and where two geminal substituents R^(a) andR^(b) together may designate optionally substituted methylene (═CH₂),and wherein two non-geminal or geminal substitutents selected fromR^(a), R^(b), and any of the substituents R^(1*), R², R^(2*), R³,R^(3*), R⁴, R⁵, R^(5*), R⁶ and R^(6*), R⁷, and R^(7*) which are presentand not involved in P, P* or the biradical(s) together may form anassociated biradical selected from biradicals of the same kind asdefined before; said pair(s) of non-geminal substituents thereby forminga mono- or bicyclic entity together with (i) the atoms to which saidnon-geminal substituents are bound and (ii) any intervening atoms; and

[0036] each of the substituents R^(1*), R², R^(2*), R³, R^(4*), R⁵,R^(5*), R⁶ and R^(6*), R⁷, and R^(7*) which are present and not involvedin P, P* or the biradical(s), is independently selected from hydrogen,optionally substituted C₁₋₁₂-alkyl, optionally substitutedC₂₋₁₂-alkenyl, optionally substituted C₂₋₁₂-alkynyl, hydroxy,C₁₋₁₂-alkoxy, C₂₋₁₂-alkenyloxy, carboxy, C₁₋₁₂-alkoxycarbonyl,C₁₋₁₂-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy,arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy,heteroarylcarbonyl, amino, mono- and di(C₁₋₆-alkyl)amino, carbamoyl,mono- and di(C₁₋₆-alkyl)-aminocarbonyl, amino-C₁₋₆-alkyl-aminocarbonyl,mono- and di(C₁₋₅-alkyl)amino-C₁₋₆-alkylaminocarbonyl,C₁₋₆-alkyl-carbonylamino, carbamido, C₁₋₆-alkanoyloxy, sulphono,C₁₋₆-alkylsulphonyloxy, nitro, azido, sulphanyl, C₁₋₆-alkylthio,halogen, DNA intercalators, photochemically active groups,thermochemically active groups, chelating groups, reporter groups, andligands, where aryl and heteroaryl may be optionally substituted, andwhere two geminal substituents together may designate oxo, thioxo,imino, or optionally substituted methylene, or together may form a spirobiradical consisting of a 1-5 carbon atom(s) alkylene chain which isoptionally interrupted and/or terminated by one or moreheteroatoms/groups selected from —O—, —S—, and —(NR^(N))— where R^(N) isselected from hydrogen and C₁₋₄-alkyl, and where two adjacent(non-geminal) substituents may designate an additional bond resulting ina double bond; and R^(N*), when present and not involved in a biradical,is selected from hydrogen and C₁₋₄-alkyl;

[0037] and basic salts and acid addition salts thereof;

[0038] with the proviso that,

[0039] (i) R³ and R⁵ do not together designate a biradical selected from—CH₂—CH₂—, —O—CH₂—, when LNA is a bicyclic nucleoside analogue;

[0040] (ii) R³, R⁵, and R⁵* do not together designate a triradical—CH₂—CH(−)—CH₂— when LNA is a tricyclic nucleoside analogue;

[0041] (iii) R^(1*) and R^(6*) do not together designate a biradical—CH₂— when LNA is a bicyclic nucleoside analogue; and

[0042] (iv) R^(4*) and R^(6*) do not together designate a biradical—CH₂—, when LNA is a bicyclic nucleoside analogue.

[0043] The present invention furthermore relates to nucleoside analogues(hereinafter LNAs) of the general formula II

[0044] wherein the substituent B is selected from nucleobases, DNAintercalators, photochemically active groups, thermochemically activegroups, chelating groups, reporter groups, and ligands;

[0045] X is selected from —O—, —S—, —N(R^(N*))—, and —C(R⁶R^(6*))—;

[0046] one of the substituents R², R^(2*), R³, and R^(3*) is a group Q*;

[0047] each of Q and Q* is independently selected from hydrogen, azido,halogen, cyano, nitro, hydroxy, Prot-O—, Act-O—, mercapto, Prot-S—,Act-S—, C₁₋₆-alkylthio, amino, Prot-N(R^(H))—, Act-N(R^(H))—, mono- ordi(C₁₋₆-alkyl)amino, optionally substituted C₁₋₆-alkoxy, optionallysubstituted C₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl, optionallysubstituted C₂₋₆-alkenyloxy, optionally substituted C₂₋₆-alkynyl,optionally substituted C₂₋₆-alkynyloxy, monophosphate, diphosphate,triphosphate, DNA intercalators, photochemically active groups,thermochemically active groups, chelating groups, reporter groups,ligands, carboxy, sulphono, hydroxymethyl, Prot-O—CH₂—, Act-O—CH₂—,aminomethyl, Prot-N(R^(H))—CH₂—, Act-N(R^(H))—CH₂—, carboxymethyl,sulphonomethyl, where Prot is a protection group for —OH, —SH, and—NH(R^(H)), respectively, Act is an activation group for —OH, —SH, and—NH(R^(H)), respectively, and R^(H) is selected from hydrogen andC₁₋₆-alkyl;

[0048] (i) R^(2*) and R^(4*) together designate a biradical selectedfrom —O—, —(CR*R*)_(r+s+1)—, —(CR*R*)_(r)—O—(CR*R*)_(s)—,—(CR*R*)_(r)—S—(CR*R*)_(s)—, —(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—,—O—(CR*R*)_(r+s)—O—, —S—(CR*R*)_(r+s)—O—, —O—(CR*R*)_(r+s)—S—,—N(R*)—(CR*R*)_(r+s)—O—, —O—(CR*R*)_(r+s)—N(R*)—, —S—(CR*R*)_(r+s)—S—,—N(R*)—(CR*R*)_(r+s)—N(R*)—, —N(R*)—(CR*R*)_(r+s)—S—, and—S—(CR*R*)_(r+s)—N(R*)—;

[0049] (ii) R² and R³ together designate a biradical selected from —O—,—(CR*R*)_(r+s)—, —(CR*R*)_(r)—O—(CR*R*)_(s)—,—(CR*R*)_(r)—S—(CR*R*)_(s)—, and —(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—;

[0050] (iii) R^(2*) and R³ together designate a biradical selected from—O—, —(CR*R*)_(r+s)—, —(CR*R*)_(r)—O—(CR*R*)_(s)—,—(CR*R*)_(r)—S—(CR*R*)_(s)—, and —(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—,

[0051] (iv) R³ and R^(4*) together designate a biradical selected from—(CR*R*)_(r)—O—(CR*R*)_(s)—, —(CR*R*)_(r)—S—(CR*R*)_(s)—, and—(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—;

[0052] (v) R³ and R⁵ together designate a biradical selected from—(CR*R*)_(r)—O—(CR*R*)_(s)—, —(CR*R*)_(r)—S—(CR*R*)_(s)—, and—(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—; or

[0053] (vi) R^(1*) and R^(4*) together designate a biradical selectedfrom —(CR*R*)_(r)—O—(CR*R*)_(s)—, —(CR*R*)_(r)—S—(CR*R*)_(s)—, and—(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—;

[0054] (vii) R^(1*) and R^(2*) together designate a biradical selectedfrom —(CR*R*)_(r)—O—(CR*R*)_(s)—, —(CR*R*)_(r)—S—(CR*R*)_(s)—, and—(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—;

[0055] wherein each R* is independently selected from hydrogen, halogen,azido, cyano, nitro, hydroxy, mercapto, amino, mono- ordi(C₁₋₆-alkyl)amino, optionally substituted C₁₋₆-alkoxy, optionallysubstituted C₁₋₆-alkyl, DNA intercalators, photochemically activegroups, thermochemically active groups, chelating groups, reportergroups, and ligands, and/or two adjacent (non-geminal) R* may togetherdesignate a double bond, and each of r and s is 0-3 with the provisothat the sum r+s is 1-4;

[0056] each of the substituents R^(1*), R², R^(2*), R³, R^(4*), R⁵, andR^(5*), which are not involved in Q, Q* or the biradical, isindependently selected from hydrogen, optionally substitutedC₁₋₁₂-alkyl, optionally substituted C₂₋₁₂-alkenyl, optionallysubstituted C₂₋₁₂-alkynyl, hydroxy, C₁₋₁₂-alkoxy, C₂₋₁₂-alkenyloxy,carboxy, C₁₋₁₂-alkoxycarbonyl, C₁₋₁₂-alkylcarbonyl, formyl, aryl,aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di(C₁₋₆-alkyl)amino, carbamoyl, mono- anddi(C₁₋₆-alkyl)-amino-carbonyl, amino-C₁₋₆-alkylaminocarbonyl, mono- anddi(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl, C₁₋₆-alkylcarbonylamino,carbamido, C₁₋₆-alkanoyloxy, sulphono, C₁₋₆-alkylsulphonyloxy, nitro,azido, sulphanyl, C₁₋₆-alkylthio, halogen, DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands, where aryl and heteroaryl may beoptionally substituted, and where two geminal substituents together maydesignate oxo, thioxo, imino, or optionally substituted methylene, ortogether may form a spiro biradical consisting of a 1-5 carbon atom(s)alkylene chain which is optionally interrupted and/or terminated by oneor more heteroatoms/groups selected from —O—, —S—, and —(NR^(N))— whereR^(N) is selected from hydrogen and C₁₋₄-alkyl, and where two adjacent(non-geminal) substituents may designate an additional bond resulting ina double bond; and R^(N*), when present and not involved in a biradical,is selected from hydrogen and C₁₋₄-alkyl;

[0057] and basic salts and acid addition salts thereof;

[0058] with the first proviso that,

[0059] (i) R³ and R⁵ do not together designate a biradical selected from—CH₂—CH₂—, —O—CH₂—, and —O—Si(^(i)Pr)₂—O—Si(Pr)₂—O—;

[0060] and with the second proviso that any chemical group (includingany nucleobase), which is reactive under the conditions prevailing inoligonucleotide synthesis, is optionally functional group protected.

[0061] The present invention also relates to the use of the nucleosideanalogues (LNAs) for the preparation of oligomers, and the use of theoligomers as well as the nucleoside analogues (LNAs) in diagnostics,molecular biology research, and in therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

[0062]FIGS. 1A and 1B illustrate known conformationally restrictednucleotides.

[0063]FIG. 2 illustrates nucleotide/nucleoside analogues of theinvention.

[0064]FIG. 3 illustrates the performance of LNA modifiedoligonucleotides in the sequence specific capture of PCR amplicons.

[0065]FIGS. 4A and 4B illustrate that LNA modified oligonucleotides areable to capture its cognate PCR amplicon by strand invasion.

[0066]FIG. 5 illustrates that LNA modified oligonucleotides, immobilisedon a solid surface, function efficiently in the sequence specificcapture of a PCR amplicon.

[0067]FIG. 6 illustrates that LNA modified oligonucleotides can act assubstrates for T4 polynucleotide kinase.

[0068]FIG. 7 illustrates that LNA modified oligonucleotides can functionas primers for nucleic acid polymerases.

[0069]FIG. 8 illustrates that LNA modified oligonucleotides canfunctions as primers in target amplification processes.

[0070]FIG. 9 illustrates that LNA modified oligonucleotides carrying a5′ anthraquinone can be covalently immobilised on a solid support byirradiation and that the immobilised oligomer is efficient in thecapture of a complementary DNA oligo.

[0071]FIG. 10 illustrates that LNA-thymidine-5′-triphosphate (LNA-TTP)can act as a substrate for terminal deoxynucleotidyl transferase (TdT).

[0072]FIG. 11 illustrates hybridisation and detection on an array withdifferent LNA modified Cy3-labelled 8 mers.

[0073]FIGS. 12 and 13 illustrate hybridisation and detection of endmismatches on an array with LNA modified Cy3-labelled 8 mers.

[0074]FIG. 14 illustrates blockade by LNA of [D-Ala2]deltorphin-inducedantinociception in the warm water tail flick test in conscious rats.

[0075]FIGS. 15A, 15B, and 15C illustrate Hybridization and detection ofend mismatches on an array with AT and all LNA modified Cy3-labelled 8mers.

[0076]FIGS. 16 and 17 illustrate that LNA can be delivered to livinghuman MCF-7 breast cancer cells.

[0077]FIGS. 18 and 19 illustrate the use of [α³³P] ddNTP's andThermoSequenase™ DNA Polymerase to sequence DNA templates containing LNAT monomers.

[0078]FIGS. 20 and 21 illustrate that exonuclease free Klenow fragmentDNA polymerase I can incorporate LNA Adenosine, Cytosine, Guanosine andUridine-5′-triphosphates into a DNA strand.

[0079]FIG. 22 illustrates the ability of terminal deoxynucleotidyltransferase (TdT) to tail LNA modified oligonucleotides.

[0080]FIGS. 23A and 23B illustrate that fully mixed LNA monomers can beused to significantly increase the performance of immobilisedbiotinylated-DNA oligos in the sequence specific capture of PCRamplicons.

[0081] FIGS. 24 to 41 illustrates possible synthetic routes towards theLNA monomers of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0082] When used herein, the term “LNA” (Locked Nucleoside Analogues)refers to the bi- and tri-cyclic nucleoside analogues of the invention,either incorporated in the oligomer of the invention (general formula I)or as discrete chemical species (general formula II). The term“monomeric LNA” specifically refers to the latter case.

[0083] Oligomers and Nucleoside Analogues

[0084] As mentioned above, the present invention i.a. relates to noveloligomers (oligonucleotides) comprising one or more bi-, tri-, orpolycyclic nucleoside analogues (hereinafter termed “LNA”). It has beenfound that the incorporation of such LNAs in place of, or as asupplement to, e.g., known nucleosides confer interesting and highlyuseful properties to an oligonucleotide. Bi- and tricyclic, especiallybicyclic, LNAs seem especially interesting within the scope of thepresent invention.

[0085] Each of the possible LNAs incorporated in an oligomer(oligonucleotide) has the general formula I

[0086] wherein X is selected from —O— (the furanose motif), —S—,—N(R^(N*)), —C(R⁶R^(6*))—, —O—C(R⁷R^(7*))—, —C(R⁶R^(6*))—O—,—S—C(R⁷R^(7*))—, —C(R⁶R^(6*))—S—, —N(R^(N*)) C(R⁷R^(7*))—,—(R⁶R⁶*)—N(R^(N*))—, and —C(R⁶R^(6*))—C(R⁷R^(7*))—, where R⁶, R^(6*),R⁷, R^(7*), and R^(N*) are as defined further below. Thus, the LNAsincorporated in the oligomer may comprise an either 5- or 6-memberedring as an essential part of the bi-, tri-, or polycyclic structure. Itis believed that 5-membered rings (X═—O—, —S—, —N(R^(N*)),—C(R⁶R^(6*))—) are especially interesting in that they are able tooccupy essentially the same conformations (however locked by theintroduction of one or more biradicals (see below)) as the nativefuranose ring of a naturally occurring nucleoside. Among the possible5-membered rings, the situations where X designates —O—, —S—, and—N(R^(N*)) seem especially interesting, and the situation where X is —O—appears to be particularly interesting.

[0087] The substituent B may designate a group which, when the oligomeris complexing with DNA or RNA, is able to interact (e.g. by hydrogenbonding or covalent bonding or electronic interaction) with DNA or RNA,especially nucleobases of DNA or RNA. Alternatively, the substituent Bmay designate a group which acts as a label or a reporter, or thesubstituent B may designate a group (e.g. hydrogen) which is expected tohave little or no interactions with DNA or RNA. Thus, the substituent Bis preferably selected from hydrogen, hydroxy, optionally substitutedC₁₋₄-alkoxy, optionally substituted C₁₋₄-alkyl, optionally substitutedC₁₋₄-acyloxy, nucleobases, DNA intercalators, photochemically activegroups, thermochemically active groups, chelating groups, reportergroups, and ligands.

[0088] In the present context, the terms “nucleobase” covers naturallyoccurring nucleobases as well as non-naturally occurring nucleobases. Itshould be clear to the person skilled in the art that variousnucleobases which previously have been considered “non-naturallyoccurring” have subsequently been found in nature. Thus, “nucleobase”includes not only the known purine and pyrimidine heterocycles, but alsoheterocyclic analogues and tautomers thereof. Illustrative examples ofnucleobases are adenine, guanine, thymine, cytosine, uracil, purine,xanthine, diaminopurine, 8-oxo-N⁶-methyladenine, 7-deazaxanthine,7-deazaguanine, N⁴,N⁴-ethanocytosin, N⁶,N⁶-ethano-2,6-diaminopurine,5-methylcytosine, 5-(C³-C⁶)-alkynylcytosine, 5-fluorouracil,5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin,isocytosine, isoguanin, inosine and the “non-naturally occurring”nucleobases described in Benner et al., U.S. Pat. No. 5,432,272. Theterm “nucleobase” is intended to cover every and all of these examplesas well as analogues and tautomers thereof. Especially interestingnucleobases are adenine, guanine, thymine, cytosine, and uracil, whichare considered as the naturally occurring nucleobases in relation totherapeutic and diagnostic application in humans.

[0089] When used herein, the term “DNA intercalator” means a group whichcan intercalate into a DNA or RNA helix, duplex or triplex. Examples offunctional parts of DNA intercalators are acridines, anthracene,quinones such as anthraquinone, indole, quinoline, isoquinoline,dihydroquinones, anthracyclines, tetracyclines, methylene blue,anthracyclinone, psoralens, coumarins, ethidium-halides, dynemicin,metal complexes such as 1,10-phenanthroline-copper,tris(4,7-diphenyl-1,10-phenanthroline)ruthenium-cobalt-enediynes such ascalcheamicin, porphyrins, distamycin, netropcin, viologen, daunomycin.Especially interesting examples are acridines, quinones such asanthraquinone, methylene blue, psoralens, coumarins, andethidium-halides.

[0090] In the present context, the term “photochemically active groups”covers compounds which are able to undergo chemical reactions uponirradiation with light. Illustrative examples of functional groupshereof are quinones, especially 6-methyl-1,4-naphtoquinone,anthraquinone, naphtoquinone, and 1,4-dimethyl-anthraquinone,diazirines, aromatic azides, benzophenones, psoralens, diazo compounds,and diazirino compounds.

[0091] In the present context “thermochemically reactive group” isdefined as a functional group which is able to undergothermochemically-induced covalent bond formation with other groups.Illustrative examples of functional parts thermochemically reactivegroups are carboxylic acids, carboxylic acid esters such as activatedesters, carboxylic acid halides such as acid fluorides, acid chlorides,acid bromide, and acid iodides, carboxylic acid azides, carboxylic acidhydrazides, sulfonic acids, sulfonic acid esters, sulfonic acid halides,semicarbazides, thiosemicarbazides, aldehydes; ketones, primaryalkohols, secondary alkohols, tertiary alkohols, phenols, alkyl halides,thiols, disulphides, primary amines, secondary amines, tertiary amines,hydrazines, epoxides, maleimides, and boronic acid derivatives.

[0092] In the present context, the term “chelating group” means amolecule that contains more than one binding site and frequently bindsto another molecule, atom or ion through more than one binding site atthe same time. Examples of functional parts of chelating groups areiminodiacetic acid, nitrilotriacetic acid, ethylenediamine tetraaceticacid (EDTA), aminophosphonic acid, etc.

[0093] In the present context, the term “reporter group” means a groupwhich is detectable either by itself or as a part of an detectionseries. Examples of functional parts of reporter groups are biotin,digoxigenin, fluorescent groups (groups which are able to absorbelectromagnetic radiation, e.g. light or X-rays, of a certainwavelength, and which subsequently reemits the energy absorbed asradiation of longer wavelength; illustrative examples are dansyl(5-dimethylamino)-1-naphthalenesulfonyl), DOXYL(N-oxyl-4,4-dimethyloxazolidine), PROXYL(N-oxyl-2,2,5,5-tetramethylpyrrolidine),TEMPO(N-oxyl-2,2,6,6-tetramethylpiperidine), dinitrophenyl, acridines,coumarins, Cy3 and Cy5 (trademarks for Biological Detection Systems,Inc.), erytrosine, coumaric acid, umbelliferone, texas red, rhodamine,tetramethyl rhodamine, Rox, 7-nitrobenzo-2-oxa-1-diazole (NBD), pyrene,fluorescein, Europium, Ruthenium, Samarium, and other rare earthmetals), radioisotopic labels, chemiluminescence labels (labels that aredetectable via the emission of light during a chemical reaction), spinlabels (a free radical (e.g. substituted organic nitroxides) or otherparamagnetic probes (e.g. Cu²⁺, Mg²⁺) bound to a biological moleculebeing detectable by the use of electron spin resonance spectroscopy),enzymes (such as peroxidases, alkaline phosphatases, β-galactosidases,and glycose oxidases), antigens, antibodies, haptens (groups which areable to combine with an antibody, but which cannot initiate an immuneresponse by itself, such as peptides and steroid hormones), carriersystems for cell membrane penetration such as: fatty acid residues,steroid moieties (cholesteryl), vitamin A, vitamin D, vitamin E, folicacid peptides for specific receptors, groups for mediating endocytose,epidermal growth factor (EGF), bradykinin, and platelet derived growthfactor (PDGF). Especially interesting examples are biotin, fluorescein,Texas Red, rhodamine, dinitrophenyl, digoxigenin, Ruthenium, Europium,Cy5, Cy3, etc.

[0094] In the present context “ligand” means something which binds.Ligands can comprise functional groups such as: aromatic groups (such asbenzene, pyridine, naphtalene, anthracene, and phenanthrene),heteroaromatic groups (such as thiophene, furan, tetrahydrofuran,pyridine, dioxane, and pyrimidine), carboxylic acids, carboxylic acidesters, carboxylic acid halides, carboxylic acid azides, carboxylic acidhydrazides, sulfonic acids, sulfonic acid esters, sulfonic acid halides,semicarbazides, thiosemicarbazides, aldehydes, ketones, primaryalcohols, secondary alcohols, tertiary alcohols, phenols, alkyl halides,thiols, disulphides, primary amines, secondary amines, tertiary amines,hydrazines, epoxides, maleimides, C₁-C₂₀ alkyl groups optionallyinterrupted or terminated with one or more heteroatoms such as oxygenatoms, nitrogen atoms, and/or sulphur atoms, optionally containingaromatic or mono/polyunsaturated hydrocarbons, polyoxyethylene such aspolyethylene glycol, oligo/polyamides such as poly-β-alanine,polyglycine, polylysine, peptides, oligo/polysaccharides,oligo/polyphosphates, toxins, antibiotics, cell poisons, and steroids,and also “affinity ligands”, i.e. functional groups or biomolecules thathave a specific affinity for sites on particular proteins, antibodies,poly- and oligosaccharides, and other biomolecules.

[0095] It will be clear for the person skilled in the art that theabove-mentioned specific examples under DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands correspond to the“active/functional” part of the groups in question. For the personskilled in the art it is furthermore clear that DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands are typically represented in theform M-K— where M is the “active/functional” part of the group inquestion and where K is a spacer through which the “active/functional”part is attached to the 5- or 6-membered ring. Thus, it should beunderstood that the group B, in the case where B is selected from DNAintercalators, photochemically active groups, thermochemically activegroups, chelating groups, reporter groups, and ligands, has the formM-K—, where M is the “active/functional” part of the DNA intercalator,photochemically active group, thermochemically active group, chelatinggroup, reporter group, and ligand, respectively, and where K is anoptional spacer comprising 1-50 atoms, preferably 1-30 atoms, inparticular 1-15 atoms, between the 5- or 6-membered ring and the“active/functional” part.

[0096] In the present context, the term “spacer” means athermochemically and photochemically non-active distance-making groupand is used to join two or more different moieties of the types definedabove. Spacers are selected on the basis of a variety of characteristicsincluding their hydrophobicity, hydrophilicity, molecular flexibilityand length (e.g. see Hermanson et. al., “Immobilized Affinity LigandTechniques”, Academic Press, San Diego, Calif. (1992), p. 137-ff).Generally, the length of the spacers are less than or about 400 Å, insome applications preferably less than 100 Å. The spacer, thus,comprises a chain of carbon atoms optionally interrupted or terminatedwith one or more heteroatoms, such as oxygen atoms, nitrogen atoms,and/or sulphur atoms. Thus, the spacer K may comprise one or more amide,ester, amino, ether, and/or thioether functionalities, and optionallyaromatic or mono/polyunsaturated hydrocarbons, polyoxyethylene such aspolyethylene glycol, oligo/polyamides such as poly-β-alanine,polyglycine, polylysine, and peptides in general, oligosaccharides,oligo/polyphosphates. Moreover the spacer may consist of combined unitsthereof. The length of the spacer may vary, taking into considerationthe desired or necessary positioning and spatial orientation of the“active/functional” part of the group in question in relation to the 5-or 6-membered ring. In particularly interesting embodiments, the spacerincludes a chemically cleavable group. Examples of such chemicallycleavable groups include disulphide groups cleavable under reductiveconditions, peptide fragments cleavable by peptidases, etc.

[0097] In one embodiment of the present invention, K designates a singlebond so that the “active/functional” part of the group in question isattached directly to the 5- or 6-membered ring.

[0098] In a preferred embodiment, the substituent B in the generalformulae I and II is preferably selected from nucleobases, in particularfrom adenine, guanine, thymine, cytosine and urasil.

[0099] In the oligomers of the present invention (formula I), Pdesignates the radical position for an internucleoside linkage to asucceeding monomer, or a 5′-terminal group. The first possibilityapplies when the LNA in question is not the 5′-terminal “monomer”,whereas the latter possibility applies when the LNA in question is the5′-terminal “monomer”. It should be understood (which also will be clearfrom the definition of internucleoside linkage and 5′-terminal groupfurther below) that such an internucleoside linkage or 5′-terminal groupmay include the substituent R⁵ (or equally applicable: the substituentR^(5*)) thereby forming a double bond to the group P. (5′-Terminalrefers to the position corresponding to the 5′ carbon atom of a ribosemoiety in a nucleoside.)

[0100] On the other hand, an internucleoside linkage to a precedingmonomer or a 3′-terminal group (P*) may originate from the positionsdefined by one of the substituents R², R^(2*), R³, and R^(3*),preferably from the positions defined by one of the substituents R³ andR^(3*). Analogously, the first possibility applies where the LNA inquestion is not the 3′-terminal “monomer”, whereas the latterpossibility applies when the LNA in question is the 3′-terminal“monomer”. (3′-Terminal refers to the position corresponding to the 3′carbon atom of a ribose moiety in a nucleoside.)

[0101] In the present context, the term “monomer” relates to naturallyoccurring nucleosides, non-naturally occurring nucleosides, PNAs, etc.as well as LNAs. Thus, the term “succeeding monomer” relates to theneighbouring monomer in the 5′-terminal direction and the “precedingmonomer” relates to the neighbouring monomer in the 3′-terminaldirection. Such succeeding and preceding monomers, seen from theposition of an LNA monomer, may be naturally occurring nucleosides ornon-naturally occurring nucleosides, or even further LNA monomers.

[0102] Consequently, in the present context (as can be derived from thedefinitions above), the term “oligomer” means an oligonucleotidemodified by the incorporation of one or more LNA(s).

[0103] The crucial part of the present invention is the presence of oneor more rings fused to the 5- or 6-membered ring illustrated with thegeneral formula I. Thus, one or two pairs of non-geminal substituentsselected from the present substituents of R^(1*), R^(4*), R⁵, R^(5*),R⁶, R^(6*), R⁷, R^(7*), R^(N*), and the ones of R², R^(2*), R³, andR^(3*) not designating P* each designates a biradical consisting of 1-8groups/atoms, preferably 1-4 groups/atoms, independently selected from—C(R^(a)R^(b))—, —C(R^(a))═C(R^(a))—, —C(R^(a))═N—, —O—, —Si(R^(a))₂—,—S—, —SO₂—, —N(R^(a))—, and >C=Z. (The term “present” indicates that theexistence of some of the substituents, i.e. R⁶, R^(6*), R⁷, R^(7*),R^(N*), is dependent on whether X includes such substituents.)

[0104] In the groups constituting the biradical(s), Z is selected from—O—, —S—, and —N(R^(a))—, and R^(a) and R^(b) each is independentlyselected from hydrogen, optionally substituted C₁₋₁₂-alkyl, optionallysubstituted C₂₋₁₂-alkenyl, optionally substituted C₂₋₁₂-alkynyl,hydroxy, C₁₋₁₂-alkoxy, C₂₋₁₂-alkenyloxy, carboxy, C₁₋₁₂-alkoxycarbonyl,C₁₋₁₂-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy,arylcarbonyl, heteroaryl, heteroaryloxycarbonyl, heteroaryloxy,heteroarylcarbonyl, amino, mono- and di(C₁₋₆-alkyl)amino, carbamoyl,mono- and di(C₁₋₆-alkyl)-amino-carbonyl, amino-C₁₋₆-alkyl-aminocarbonyl,mono- and di(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl,C₁₋₆-alkyl-carbonylamino, carbamido, C₁₋₆-alkanoyloxy, sulphono,C₁₋₆-alkylsulphonyloxy, nitro, azido, sulphanyl, C₁₋₆-alkylthio,halogen, DNA intercalators, photochemically active groups,thermochemically active groups, chelating groups, reporter groups, andligands (where the latter groups may include a spacer as defined for thesubstituent B), where aryl and heteroaryl may be optionally substituted.Moreover, two geminal substituents R^(a) and R^(b) together maydesignate optionally substituted methylene (═CH₂ optionally substitutedone or two times with substituents as defined as optional substituentsfor aryl), and two non-geminal or geminal substituents selected fromR^(a), R^(b), and any of the substituents R^(1*), R², R^(2*), R³,R^(3*), R⁴, R⁵, R^(5*), R⁶ and R^(6*), R⁷, and R^(7*) which are presentand not involved in P, P* or the biradical(s) may together form anassociated biradical selected from biradicals of the same kind asdefined before. It will be clear that each of the pair(s) of non-geminalsubstituents thereby forms a mono- or bicyclic entity together with (i)the atoms to which the non-geminal substituents are bound and (ii) anyintervening atoms.

[0105] It is believed that biradicals which are bound to the ring atomsof the 5- or 6-membered rings are preferred in that inclusion of thesubstituents R⁵ and R^(5*) may cause an undesired sterical interactionwith internucleoside linkage. Thus, it is preferred that the one or twopairs of non-geminal substituents, which are constituting one or twobiradical(s), respectively, are selected from the present substituentsof R^(1*), R^(4*), R⁶, R^(6*), R⁷, R^(7*), R^(N*), and the ones of R²,R^(2*), R³, and R^(3*) not designating P*.

[0106] Preferably, the LNAs incorporated in the oligomers comprise onlyone biradical constituted by a pair of (two) non-geminal substituents.In particular, it is preferred that R^(3*) designates P* and that thebiradical is formed between R^(2*) and R^(4*) or R² and R³.

[0107] This being said, it should be understood (especially with dueconsideration of the known bi- and tricyclic nucleoside analogues—see“Background of the Invention”) that the present invention does notrelate to oligomers comprising the following bi- or tricyclicnucleosides analogues:

[0108] (i) R² and R³ together designate a biradical selected from—O—CH₂—CH₂— and —O—CH₂—CH₂—CH₂— when LNA is a bicyclic nucleosideanalogue;

[0109] (ii) R³ and R⁵ together designate a biradical selected from—CH₂—CH₂—, —O—CH₂—, when LNA is a bicyclic nucleoside analogue;

[0110] (iii) R³, R⁵, and R⁵* together designate a triradical—CH₂—CH(−)—CH₂— when LNA is a tricyclic nucleoside analogue;

[0111] (iv) R^(1*) and R^(6*) together designate a biradical —CH₂— whenLNA is a bicyclic nucleoside analogue; or

[0112] (v) R^(4*) and R^(6*) together designate a biradical —CH₂— whenLNA is a bicyclic nucleoside analogue;

[0113] except where such bi- or tricyclic nucleoside analogues arecombined with one or more of the novel LNAs defined herein.

[0114] In the present context, i.e. in the present description andclaims, the orientation of the biradicals are so that the left-hand siderepresents the substituent with the lowest number and the right-handside represents the substituent with the highest number, thus, when R³and R⁵ together designate a biradical “—O—CH₂—”, it is understood thatthe oxygen atom represents R³, thus the oxygen atom is e.g. attached tothe position of R³, and the methylene group represents R⁵.

[0115] Considering the numerous interesting possibilities for thestructure of the biradical(s) in LNA(s) incorporated in oligomersaccording to the invention, it is believed that the biradical(s)constituted by pair(s) of non-geminal substituents preferably is/areselected from —(CR*R*)_(r)—Y—(CR*R*)_(s)—,—(CR*R*)_(r)—Y—(CR*R*)_(s)—Y—, —Y—(CR*R*)_(r+s)—Y—,—Y—(CR*R*)_(r)—Y—(CR*R*)_(s)—, —(CR*R*)_(r+s), —Y—, —Y—Y—, wherein eachY is independently selected from —O—, —S—, —Si(R*)₂—, —N(R*)—, >C═O,—C(═O)—N(R*)—, and —N(R*)—C(═O)—, each R* is independently selected fromhydrogen, halogen, azido, cyano, nitro, hydroxy, mercapto, amino, mono-or di(C₁₋₆-alkyl)amino, optionally substituted C₁₋₆-alkoxy, optionallysubstituted C₁₋₆-alkyl, DNA intercalators, photochemically activegroups, thermochemically active groups, chelating groups, reportergroups, and ligands, and/or two adjacent (non-geminal) R* may togetherdesignate a double bond; and each of r and s is 0-4 with the provisothat the sum r+s is 1-5. Particularly interesting situations are thosewherein each biradical is independently selected from —Y—,—(CR*R*)_(r+s)—, —(CR*R*)_(r)—Y—(CR*R*)_(s)—, and —Y—(CR*R*)_(r+s)—Y—,wherein and each of r and s is 0-3 with the proviso that the sum r+s is1-4.

[0116] Considering the positioning of the biradical in the LNA(s), it isbelieved (based on the preliminary findings (see the examples)) that thefollowing situations are especially interesting, namely where: R^(2*)and R^(4*) together designate a biradical selected from —Y—,—(CR*R*)_(r+s+1)—, —(CR*R*)_(r)—Y—(CR*R*)_(s)—, and —Y—(CR*R*)_(r+s)—Y—;R² and R³ together designate a biradical selected from —Y—,—(CR*R*)_(r+s)—, —(CR*R*)_(r)—Y—(CR*R*)_(s)—, and —Y—(CR*R*)_(r+s)—Y—;R^(2*) and R³ together designate a biradical selected from —Y—,—(CR*R*)_(r+s)—, —(CR*R*)_(r)—Y—(CR*R*)_(s)—, and —Y—(CR*R*)_(r+s)—Y—;R³ and R^(4*) together designate a biradical selected from —Y—,—(CR*R*)_(r+s)—, —(CR*R*)_(r)—Y—(CR*R*)_(s)—, and —Y—(CR*R*)_(r+s)—Y—;R³ and R⁵ together designate a biradical selected from —Y′—,—(CR*R*)_(r+s+1)—, —(CR*R*)_(r)—Y—(CR*R*)_(s)—, and —Y—(CR*R*)_(r+s)—Y—;R^(1*) and R^(4*) together designate a biradical selected from —Y′—,—(CR*R*)_(r+s+1)—, —(CR*R*)_(r)—Y—(CR*R*)_(s)—, and—Y—(CR*R*)_(r+s)NR*—; or where R^(1*) and R^(2*) together designate abiradical selected from —Y—, —(CR*R*)_(r+s)—,—(CR*R*)_(r)—Y—(CR*R*)_(s)—, and —Y—(CR*R*)_(r+s)—Y—; wherein each of rand s is 0-3 with the proviso that the sum r+s is 1-4, Y is as definedabove, and where Y′ is selected from —NR*—C(═O)— and —C(═O)—NR*—.

[0117] Particularly interesting oligomers are those wherein one of thefollowing criteria applies for at least one LNA in an oligomer: R^(2*)and R^(4*) together designate a biradical selected from —O—, —S—,—N(R*)—, —(CR*R*)_(r+s+1)—, —(CR*R*)_(r)—O—(CR*R*)_(s)—,—(CR*R*)_(r)—S—(CR*R*)_(s)—, —(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—,—O—(CR*R*)_(r+s)—O—, —S—(CR*R*)_(r+s)—O—, —O—(CR*R*)_(r+s)—S—,—N(R*)—(CR*R*)_(r+s)—O—, —O—(CR*R*)_(r+s)—N(R*)—, —S—(CR*R*)_(r+s)—S—,—N(R*)—(CR*R*)_(r+s)—N(R*)—, —N(R*)—(CR*R*)_(r+s)—S—, and—S—(CR*R*)_(r+s)—N(R*)—; R² and R³ together designate a biradicalselected from —O—, —(CR*R*)_(r+s)—, —(CR*R*)_(r)—O—(CR*R*)_(s)—,—(CR*R*)_(r)—S—(CR*R*)_(s)—, and —(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—; R^(2*)and R³ together designate a biradical selected from —O—,—(CR*R*)_(r+s)—, —(CR*R*)_(r)—O—(CR*R*)_(s)—,—(CR*R*)_(r)—S—(CR*R*)_(s)—, and —(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—; R³ andR^(4*) together designate a biradical selected from—(CR*R*)_(r)—O—(CR*R*)_(s)—, —(CR*R*)_(r)—S—(CR*R*)_(s)—, and—(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—; R³ and R⁵ together designate abiradical selected from —(CR*R*)_(r)—O—(CR*R*)_(s)—,—(CR*R*)_(r)—S—(CR*R*)_(s)—, and —(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—; R^(1*)and R^(4*) together designate a biradical selected from—(CR*R*)_(r)—O—(CR*R*)_(s)—, —(CR*R*)_(r)—S—(CR*R*)_(s)—, and—(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—; or R^(1*) and R^(2*) together designatea biradical selected from —(CR*R*)_(r)—O—(CR*R*)_(s)—,—(CR*R*)_(r)—S—(CR*R*)_(s)—, and —(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—;wherein each of r and s is 0-3 with the proviso that the sum r+s is 1-4,and where R^(H) designates hydrogen or C₁₋₄-alkyl.

[0118] It is furthermore preferred that one R* is selected fromhydrogen, hydroxy, optionally substituted C₁₋₆-alkoxy, optionallysubstituted C₁₋₆-alkyl, DNA intercalators, photochemically activegroups, thermochemically active groups, chelating groups, reportergroups, and ligands, and any remaining substituents R* are hydrogen.

[0119] In one preferred embodiment, one group R* in the biradical of atleast one LNA is selected from DNA intercalators, photochemically activegroups, thermochemically active groups, chelating groups, reportergroups, and ligands (where the latter groups may include a spacer asdefined for the substituent B).

[0120] With respect to the substituents R^(1*), R², R^(2*), R³, R^(4*),R⁵, R^(5*), R⁶ and R^(6*), R⁷, and R^(7*), which are present and notinvolved in P, P* or the biradical(s), these are independently selectedfrom hydrogen, optionally substituted C₁₋₁₂-alkyl, optionallysubstituted C₂₋₁₂-alkenyl, optionally substituted C₂₋₁₂-alkynyl,hydroxy, C₁₋₁₂-alkoxy, C₂₋₁₂-alkenyloxy, carboxy, C₁₋₁₂-alkoxycarbonyl,C₁₋₁₂-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy,arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy,heteroarylcarbonyl, amino, mono- and di(C₁₋₆-alkyl)amino, carbamoyl,mono- and di(C₁₋₆-alkyl)-amino-carbonyl, amino-C₁₋₆-alkyl-aminocarbonyl,mono- and di(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl,C₁₋₆-alkyl-carbonylamino, carbamido, C₁₋₆-alkanoyloxy, sulphono,C₁₋₆-alkylsulphonyloxy, nitro, azido, sulphanyl, C₁₋₆-alkylthio,halogen, DNA intercalators, photochemically active groups,thermochemically active groups, chelating groups, reporter groups, andligands (where the latter groups may include a spacer as defined for thesubstituent B), where aryl and heteroaryl may be optionally substituted,and where two geminal substituents together may designate oxo, thioxo,imino, or optionally substituted methylene, or together may form a spirobiradical consisting of a 1-5 carbon atom(s) alkylene chain which isoptionally interrupted and/or terminated by one or moreheteroatoms/groups selected from —O—, —S—, and —(NR^(N))— where R^(N) isselected from hydrogen and C₁₋₄-alkyl, and where two adjacent(non-geminal) substituents may designate an additional bond resulting ina double bond; and R^(N*), when present and not involved in a biradical,is selected from hydrogen and C₁₋₄-alkyl.

[0121] Preferably, each of the substituents R^(1*), R², R^(2*), R³,R^(3*), R^(4*), R⁵, R^(5*), R⁶, R^(6*), R⁷, and R^(7*) of the LNA(s),which are present and not involved in P, P* or the biradical(s), isindependently selected from hydrogen, optionally substituted C₁₋₆-alkyl,optionally substituted C₂₋₆-alkenyl, hydroxy, C₁₋₆-alkoxy,C₂₋₆-alkenyloxy, carboxy, C₁₋₆-alkoxycarbonyl, C₁₋₆-alkylcarbonyl,formyl, amino, mono- and di(C₁₋₆-alkyl)amino, carbamoyl, mono- anddi(C₁₋₆-alkyl)-amino-carbonyl, C₁₋₆-alkyl-carbonylamino, carbamido,azido, C₁₋₆-alkanoyloxy, sulphono, sulphanyl, C₁₋₆-alkylthio, DNAintercalators, photochemically active groups, thermochemically activegroups, chelating groups, reporter groups, and ligands, and halogen,where two geminal substituents together may designate oxo, and whereR^(N*), when present and not involved in a biradical, is selected fromhydrogen and C₁₋₄-alkyl.

[0122] In a preferred embodiment of the present invention, X is selectedfrom —O—, —S—, and —NR^(N*)—, in particular —O—, and each of thesubstituents R^(1*), R², R^(2*), R³, R^(3*), R^(4*), R⁵, R^(5*), R⁶,R^(6*), R⁷, and R^(7*) of the LNA(s), which are present and not involvedin P, P* or the biradical(s), designate hydrogen.

[0123] In an even more preferred embodiment of the present invention,R^(2*) and R^(4*) of an LNA incorporated into an oligomer togetherdesignate a biradical. Preferably, X is O, R² selected from hydrogen,hydroxy, and optionally substituted C₁₋₆-alkoxy, and R^(1*), R³, R⁵, andR^(5*) designate hydrogen, and, more specifically, the biradical isselected from —O—, —(CH₂)₀₋₁—O—(CH₂)₁₋₃—, —(CH₂)₀₋₁—S—(CH₂)₁₋₃—,—(CH₂)₀₋₁—N(R^(N))—(CH₂)₁₋₃—, and —(CH₂)₂₋₄—, in particular from—O—CH₂—, —S—CH₂—, and —NR^(H)—CH₂—. Generally, with due regard to theresults obtained so far, it is preferred that the biradical constitutingR^(2*) and R^(4*) forms a two carbon atom bridge, i.e. the biradicalforms a five membered ring with the furanose ring (X═O).

[0124] In another embodiment of the present invention, R² and R³ of anLNA incorporated into an oligomer together designate a biradical.Preferably, X is O, R^(2*) is selected from hydrogen, hydroxy, andoptionally substituted C₁₋₆-alkoxy, and R^(1*), R^(4*), R⁵, and R^(5*)designate hydrogen, and, more specifically, the biradical is selectedfrom —(CH₂)₀₋₁—O—(CH₂)₁₋₃—, —(CH₂)₀₋₁—S—(CH₂)₁₋₃—,—(CH₂)₀₋₁—N(R^(H))—(CH₂)₁₋₃— and —(CH₂)₁₋₄—, in particular from —O—CH₂—,—S—CH₂—, —N(R^(H))—CH₂—. In the latter case, the amino and thio variantsappears to be particularly interesting.

[0125] In a further embodiment of the present invention, R^(2*) and R³of an LNA incorporated into an oligomer together designate a biradical.Preferably, X is O, R² is selected from hydrogen, hydroxy, andoptionally substituted C₁₋₆-alkoxy, and R^(1*), R^(4*), R⁵, and R^(5*)designate hydrogen, and, more specifically, the biradical is selectedfrom —(CH₂)₀₋₁—O—(CH₂)₁₋₃— and —(CH₂)₂₋₄—.

[0126] In a further embodiment of the present invention, R³ and R^(4*)of an LNA incorporated into an oligomer together designate a biradical.Preferably, X is O, R^(2*) selected from hydrogen, hydroxy, andoptionally substituted C₁₋₆-alkoxy, and R^(1*), R², R⁵, and R^(5*)designate hydrogen, and, more specifically, the biradical is—(CH₂)₀₋₂—O—(CH₂)₀₋₂—.

[0127] In a further embodiment of the present invention, R³ and R^(5*)of an LNA incorporated into an oligomer together designate a biradical.Preferably, X is O, R^(2*) selected from hydrogen, hydroxy, andoptionally substituted C₁₋₆-alkoxy, and R^(1*), R², R⁴, and R⁵ designatehydrogen, and, more specifically, the biradical is selected from—O—(CHR*)₂₋₃— and —(CHR*)₁₋₃—O—(CHR*)₀₋₃—.

[0128] In a further embodiment of the present invention, R^(1*) andR^(4*) of an LNA incorporated into an oligomer together designate abiradical. Preferably, X is O, R^(2*) selected from hydrogen, hydroxy,and optionally substituted C₁₋₆-alkoxy, and R², R³, R⁵, and R^(5*)designate hydrogen, and, more specifically, the biradical is—(CH₂)₀₋₂—O—(CH₂)₀₋₂—.

[0129] In these embodiments, it is furthermore preferred that at leastone LNA incorporated in an oligomer includes a nucleobase (substituentB) selected from adenine and guanine. In particular, it is preferredthat an oligomer have LNA incorporated therein both include at least onenucleobase selected from thymine, urasil and cytosine and at least onenucleobase selected from adenine and guanine. For LNA monomers, it isespecially preferred that the nucleobase is selected from adenine andguanine.

[0130] For these interesting embodiments, it is also preferred that theLNA(s) has/have the general formula Ia (see below).

[0131] Within a variant of these interesting embodiments, all monomersof a oligonucleotide are LNA monomers.

[0132] As it will be evident from the general formula I (LNA(s) in anoligomer) (and the general formula II (monomeric LNA)—see below) and thedefinitions associated therewith, there may be one or several asymmetriccarbon atoms present in the oligomers (and monomeric LNAs) depending onthe nature of the substituents and possible biradicals, cf. below. Theoligomers prepared according to the method of the invention, as well asthe oligomers per se, are intended to include all stereoisomers arisingfrom the presence of any and all isomers of the individual monomerfragments as well as mixtures thereof, including racemic mixtures. Whenconsidering the 5- or 6-membered ring, it is, however, believed thatcertain stereochemical configurations will be especially interesting,e.g. the following

[0133] where the wavy lines represent the possibility of bothdiastereomers arising from the interchange of the two substituents inquestion.

[0134] An especially interesting stereoisomeric representation is thecase where the LNA(s) has/have the following formula Ia

[0135] Also interesting as a separate aspect of the present invention isthe variant of formula Ia where B is in the “α-configuration”.

[0136] In these cases, as well as generally, R^(3*) preferablydesignates P*.

[0137] The oligomers according to the invention typically comprise1-10000 LNA(s) of the general formula I (or of the more detailed generalformula Ia) and 0-10000 nucleosides selected from naturally occurringnucleosides and nucleoside analogues. The sum of the number ofnucleosides and the number of LNA(s) is at least 2, preferably at least3, in particular at least 5, especially at least 7, such as in the rangeof 2-15000, preferably in the range of 2-100, such as 3-100, inparticular in the range of 2-50, such as 3-50 or 5-50 or 7-50.

[0138] Preferably at least one LNA comprises a nucleobase as thesubstituent B.

[0139] In the present context, the term “nucleoside” means a glycosideof a heterocyclic base. The term “nucleoside” is used broadly as toinclude non-naturally occurring nucleosides, naturally occurringnucleosides as well as other nucleoside analogues. Illustrative examplesof nucleosides are ribonucleosides comprising a ribose moiety as well asdeoxyribonuclesides comprising a deoxyribose moiety. With respect to thebases of such nucleosides, it should be understood that this may be anyof the naturally occurring bases, e.g. adenine, guanine, cytosine,thymine, and uracil, as well as any modified variants thereof or anypossible unnatural bases.

[0140] When considering the definitions and the known nucleosides(naturally occurring and non-naturally occurring) and nucleosideanalogues (including known bi- and tricyclic analogues), it is clearthat an oligomer may comprise one or more LNA(s) (which may be identicalor different both with respect to the selection of substituent and withrespect to selection of biradical) and one or more nucleosides and/ornucleoside analogues. In the present context “oligonucleotide” means asuccessive chain of nucleosides connected via internucleoside linkages,however, it should be understood that a nucleobase in one or morenucleotide units (monomers) in an oligomer (oligonucleotide) may havebeen modified with a substituent B as defined above.

[0141] The oligomers may be linear, branched or cyclic. In the case of abranched oligomer, the branching points may be located in a nucleoside,in an internucleoside linkage or, in an intriguing embodiment, in anLNA. It is believed that in the latter case, the substituents R²,R^(2*), R³, and R^(3*) may designate two groups P* each designating aninternucleoside linkage to a preceding monomer, in particular, one of R²and R^(2*) designate P* and one or R³ and R^(3*) designate a further P*.

[0142] As mentioned above, the LNA(s) of an oligomer are connected withother monomers via an internucleoside linkage. In the present context,the term “internucleoside linkage” means a linkage consisting of 2 to 4,preferably 3, groups/atoms selected from —CH₂—, —O—, —S—,—NR^(H)—, >C═O, >C═NR^(H), >C═S, —Si(R″)₂—, —SO— —S(O)₂—, —P(O)₂—,—PO(BH₃)—, —P(O,S)—, —P(S)₂—, —PO(R″)—, —PO(OCH₃)—, and —PO(NHR^(H))—,where R^(H) is selected form hydrogen and C₁₋₄-alkyl, and R″ is selectedfrom C₁₋₆-alkyl and phenyl. Illustrative examples of suchinternucleoside linkages are —CH₂—CH₂—CH₂—, —CH₂—CO—CH₂—,—CH₂—CHOH—CH₂—, —O—CH₂—O—, —O—CH₂—CH₂—, —O—CH₂—CH═ (including R⁵ whenused as a linkage to a succeeding monomer), —CH₂—CH₂—O—,—NR^(H)—CH₂—CH₂—, —CH₂—CH₂—NR^(H), —CH₂—NR^(H)—CH₂—, —O—CH₂—CH₂—NR^(H)—,—NR^(H)—CO—O—, —NR^(H)CO—NR^(H), —NR^(H)CS—NR^(H)—,—NR^(H)—C(═NR^(H))—NR^(H), —NR^(H)—CO—CH₂—NR^(H)—, —O—CO—O—,—O—CO—CH₂—O—, —O—CH₂—CO—O—, —CH₂—CO—NR^(H), —O—CO—NR^(H)—,—NR^(H)CO—CH₂—, —O—CH₂—CO—NR^(H)—, —O—CH₂—CH₂—NR^(H)—, —CH═N—O—,—CH₂—NR^(H)—O—, —CH₂—O—N═ (including R⁵ when used as a linkage to asucceeding monomer), —CH₂—O—NR^(H)—, —CO—NR^(H)CH₂—, —CH₂—NR^(H)O,—CH₂NR^(H)—CO—, —O—NR^(H)—CH₂—, —O—NR^(H)—, —O—CH₂—S—, —S—CH₂—O—,—CH₂—CH₂—S—, —O—CH₂—CH₂—S—, —S—CH₂—CH═ (including R⁵ when used as alinkage to a succeeding monomer), —S—CH₂—CH₂—, —S—CH₂—CH₂—O—,—S—CH₂—CH₂—S—, —CH₂—S—CH₂—, —CH₂—SO—CH₂—, —CH₂—SO₂—CH₂—, —O—SO—O—,—O—S(O)₂—O—, —O—S(O)₂—CH₂—, —O—S(O)₂—NR^(H)—, —NR^(H)—S(O)₂—CH₂—,—O—S(O)₂—CH₂—, —O—P(O)₂—O—, —O—P(O,S)—O—, —O—P(S)₂—O—, —S—P(O)₂—O—,—S—P(O,S)—O—, —S—P(S)₂—O—, —O—P(O)₂—S—, —O—P(O,S)—S—, —O—P(S)₂—S—,—S—P(O)₂—S—, —S—P(O,S)—S—, —S—P(S)₂—S—, —O—PO(R″)—O—, —O—PO(OCH₃)—O—,—O—PO(OCH₂CH₃)—O—, —O—PO(OCH₂CH₂S—R)—O—, —O—PO(BH₃)—O—,—O—PO(NHR^(N))—O—, —O—P(O)₂—NR^(H)—, —NR^(H)—P(O)₂—O—,—O—P(O,NR^(H))—O—, —CH₂—P(O)₂—O—, —O—P(O)₂—CH₂—, and —O—Si(R″)₂—O—;among which CH₂CONR^(H), CH₂—NR^(H)—O—, —S—CH₂—O—, —O—P(O)₂—O—,—O—P(O,S)—O—, —O—P(S)₂—O—, NR^(H)P(O)₂—O—, —O—P(O,NR^(H))—O—,—O—PO(R″)—O—, —O—PO(CH₃)—O—, and —O—PO(NHR^(N))—O—, where R^(H) isselected form hydrogen and C₁₋₄-alkyl, and R″ is selected fromC₁₋₆-alkyl and phenyl, are especially preferred. Further illustrativeexamples are given in Mesmaeker et. al., Current Opinion in StructuralBiology 1995, 5, 343-355. The left-hand side of the internucleosidelinkage is bound to the 5- or 6-membered ring as substituent P*, whereasthe right-hand side is bound to the 5′-position of a preceding monomer.It is also clear from the above that the group P may also designate a5′-terminal group in the case where the LNA in question is the5′-terminal monomer. Examples of such 5′-terminal groups are hydrogen,hydroxy, optionally substituted C₁₋₆-alkyl, optionally substitutedC₁₋₆-alkoxy, optionally substituted C₁₋₆-alkylcarbonyloxy, optionallysubstituted aryloxy, monophosphate, diphosphate, triphosphate, and—W-A′, wherein W is selected from —O—, —S—, and —N(R^(H))— where R^(H)is selected from hydrogen and C₁₋₆-alkyl, and where A′ is selected fromDNA intercalators, photochemically active groups, thermochemicallyactive groups, chelating groups, reporter groups, and ligands (where thelatter groups may include a spacer as defined for the substituent B).

[0143] In the present description and claims, the terms “monophosphate”,“diphosphate”, and “triphosphate” mean groups of the formula:—O—P(O)₂—O⁻, —O—P(O)₂—O—P(O)₂—O⁻, and —O—P(O)₂—O—P(O)₂—O—P(O)₂—O⁻,respectively.

[0144] In a particularly interesting embodiment, the group P designatesa 5′-terminal groups selected from monophosphate, diphosphate andtriphosphate. Especially the triphosphate variant is interesting as asubstrate

[0145] Analogously, the group P* may designate a 3′-terminal group inthe case where the LNA in question is the 3′-terminal monomer. Examplesof such 3′-terminal groups are hydrogen, hydroxy, optionally substitutedC₁₋₆-alkoxy, optionally substituted C₁₋₆-alkylcarbonyloxy, optionallysubstituted aryloxy, and —W-A′, wherein W is selected from —O—, —S—, and—N(R^(H))— where R^(H) is selected from hydrogen and C₁₋₆-alkyl, andwhere A′ is selected from DNA intercalators, photochemically activegroups, thermochemically active groups, chelating groups, reportergroups, and ligands (where the latter groups may include a spacer asdefined for the substituent B).

[0146] In a preferred embodiment of the present invention, the oligomerhas the following formula V:

G-[Nu-L]_(n(0))-{([LNA-L]_(m(q))-[Nu-L]_(n(q))}_(q)-G*  V

[0147] wherein

[0148] q is 1-50;

[0149] each of n(0), . . . , n(q) is independently 0-10000;

[0150] each of m(1), . . . , m(q) is independently 1-10000;

[0151] with the proviso that the sum of n(0), . . . , n(q) and m(1), . .. , m(q) is 2-15000;

[0152] G designates a 5′-terminal group;

[0153] each Nu independently designates a nucleoside selected fromnaturally occurring nucleosides and nucleoside analogues;

[0154] each LNA independently designates a nucleoside analogue;

[0155] each L independently designates an internucleoside linkagebetween two groups selected from Nu and LNA, or L together with G*designates a 3′-terminal group; and

[0156] each LNA-L independently designates a nucleoside analogue of thegeneral formula I as defined above, or preferably of the general formulaIa as defined above.

[0157] Within this embodiment, as well as generally, the presentinvention provides the intriguing possibility of including LNAs withdifferent nucleobases, in particular both nucleobases selected fromthymine, cytosine and urasil and nucleobases selected from adenine andguanine.

[0158] In another embodiment of the present invention, the oligomerfurther comprises a PNA mono- or oligomer segment of the formula

[0159] wherein B is a defined above for the formula I, AASC designateshydrogen or an amino acid side chain, t is 1-5, and w is 1-50.

[0160] In the present context, the term “amino acid side chain” means agroup bound to the α-atom of an α-amino acids, i.e. corresponding to theα-amino acid in question without the glycine moiety, preferably aneither naturally occurring or a readily available α-amino acid.Illustrative examples are hydrogen (glycine itself), deuterium(deuterated glycine), methyl (alanine), cyanomethyl (β-cyano-alanine),ethyl, 1-propyl (norvaline), 2-propyl (valine), 2-methyl-1-propyl(leucine), 2-hydroxy-2-methyl-1-propyl (β-hydroxy-leucine), 1-butyl(norleucine), 2-butyl (isoleucine), methylthioethyl (methionine), benzyl(phenylalanine), p-amino-benzyl (p-amino-phenylalanine), p-iodo-benzyl(p-iodo-phenylalanine), p-fluoro-benzyl (p-fluoro-phenylalanine),p-bromo-benzyl (p-bromo-phenylalanine), p-chloro-benzyl(p-chloro-phenylalanine), p-nitro-benzyl (p-nitro-phenylalanine),3-pyridylmethyl (β-(3-pyridyl)-alanine), 3,5-diiodo-4-hydroxybenzyl(3,5-diiodo-tyrosine), 3,5-dibromo-4-hydroxy-benzyl(3,5-dibromo-tyrosine), 3,5-dichloro-4-hydroxy-benzyl(3,5-dichloro-tyrosine), 3,5-difluoro-4-hydroxy-benzyl(3,5-difluoro-tyrosine), 4-methoxy-benzyl (O-methyl-tyrosine),2-naphtylmethyl (β-(2-naphtyl)-alanine), 1-naphtylmethyl(β-(1-naphtyl)-alanine), 3-indolylmethyl (tryptophan), hydroxymethyl(serine), 1-hydroxyethyl (threonine), mercaptomethyl (cysteine),2-mercapto-2-propyl (penicillamine), 4-hydroxybenzyl (tyrosine),aminocarbonylmethyl (asparagine), 2-aminocarbonylethyl (glutamine),carboxymethyl (aspartic acid), 2-carboxyethyl (glutamic acid),aminomethyl (α,β-diaminopropionic acid), 2-aminoethyl(α,γ-diaminobutyric acid), 3-amino-propyl (ornithine), 4-amino-1-butyl(lysine), 3-guanidino-1-propyl (arginine), and 4-imidazolylmethyl(histidine).

[0161] PNA mono- or oligomer segment may be incorporated in a oligomeras described in EP 0672677 A2.

[0162] The oligomers of the present invention are also intended to coverchimeric oligomers. “Chimeric oligomers” means two or more oligomerswith monomers of different origin joined either directly or via aspacer. Illustrative examples of such oligomers which can be combinedare peptides, PNA-oligomers, oligomers containing LNA's, andoligonucleotide oligomers.

[0163] Apart from the oligomers defined above, the present inventionalso provides monomeric LNAs useful, e.g., in the preparation ofoligomers, as substrates for, e.g., nucleic acid polymerases,polynucleotide kinases, terminal transferases, and as therapeuticalagents, see further below. The monomeric LNAs correspond in the overallstructure (especially with respect to the possible biradicals) to theLNAs defined as constituents in oligomers, however with respect to thegroups P and P*, the monomeric LNAs differ slightly as will be explainedbelow. Furthermore, the monomeric LNAs may comprise functional groupprotecting groups, especially in the cases where the monomeric LNAs areto be incorporated into oligomers by chemical synthesis.

[0164] An interesting subgroup of the possible monomeric LNAs comprisesbicyclic nucleoside analogues (LNAs) of the general formula II

[0165] wherein the substituent B is selected from nucleobases, DNAintercalators, photochemically active groups, thermochemically activegroups, chelating groups, reporter groups, and ligands; X is selectedfrom —O—, —S—, —N(R^(N*))—, and —C(R⁶R^(6*))—, preferably from —O—, —S—,and —N(R^(N*))—; one of the substituents R², R^(2*), R³, and R^(3*) is agroup Q*;

[0166] each of Q and Q* is independently selected from hydrogen, azido,halogen, cyano, nitro, hydroxy, Prot-O—, Act-O—, mercapto, Prot-S—,Act-S—, C₁₋₆-alkylthio, amino, Prot-N(R^(H))—, Act-N(R^(H))—, mono- ordi(C₁₋₆-alkyl)amino, optionally substituted C₁₋₆-alkoxy, optionallysubstituted C₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl, optionallysubstituted C₂₋₆-alkenyloxy, optionally substituted C₂₋₆-alkynyl,optionally substituted C₂₋₆-alkynyloxy, monophosphate, diphosphate,triphosphate, DNA intercalators, photochemically active groups,thermochemically active groups, chelating groups, reporter groups,ligands, carboxy, sulphono, hydroxymethyl, Prot-O—CH₂—, Act-O—CH₂—,aminomethyl, Prot-N(R^(H))—CH₂—, Act-N(R^(H))—CH₂—, carboxymethyl,sulphonomethyl, where Prot is a protection group for —OH, —SH, and—NH(R^(H)), respectively, Act is an activation group for —OH, —SH, and—NH(R^(H)), respectively, and R^(H) is selected from hydrogen andC₁₋₆-alkyl;

[0167] R^(2*) and R^(4*) together designate a biradical selected from—O—, —S—, —N(R*)—, —(CR*R*)_(r+s+1)—, —(CR*R*)_(r)—O—(CR*R*)_(s)—,—(CR*R*)_(r)—S—(CR*R*)_(s)—, —(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—,—O—(CR*R*)_(r+s)—O—, —S—(CR*R*)_(r+s)—O—, —O—(CR*R*)_(r+s)—S—,—N(R*)—(CR*R*)_(r+s)—O—, —O—(CR*R*)_(r+s)—N(R*)—, —S—(CR*R*)_(r+s)—S—,—N(R*)—(CR*R*)_(r+s)—N(R*)—, —N(R*)—(CR*R*)_(r+s)—S—, and—S—(CR*R*)_(r+s)—N(R*)—; R² and R³ together designate a biradicalselected from —O—, —(CR*R*)_(r+s)—, —(CR*R*)_(r)—O—(CR*R*)_(s)—,—(CR*R*)_(r)—S—(CR*R*)_(s)—, and —(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—; R^(2*)and R³ together designate a biradical selected from —O—,—(CR*R*)_(r+s)—, —(CR*R*)_(r)—O—(CR*R*)_(s)—,—(CR*R*)_(r)—S—(CR*R*)_(s)—, and —(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—; R³ andR^(4*) together designate a biradical selected from—(CR*R*)_(r)—O—(CR*R*)_(s)—, —(CR*R*)_(r)—S—(CR*R*)_(s)—, and—(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—; R³ and R⁵ together designate abiradical selected from —(CR*R*)_(r)—O—(CR*R*)_(s)—,—(CR*R*)_(r)—S—(CR*R*)_(s)—, and —(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—; R^(1*)and R^(4*) together designate a biradical selected from—(CR*R*)_(r)—O—(CR*R*)_(s)—, —(CR*R*)_(r)—S—(CR*R*)_(s)—, and—(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—; or R^(1*) and R^(2*) together designatea biradical selected from —(CR*R*)_(r)—O—(CR*R*)_(s)—,—(CR*R*)_(r)—S—(CR*R*)_(s)—, and —(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—;wherein R* is as defined above for the oligomers; and each of thesubstituents R^(1*), R², R^(2*), R³, R^(4*), R⁵, and R^(5*), which arenot involved in Q, Q* or the biradical, are as defined above for theoligomers.

[0168] It should furthermore be understood, with due consideration ofthe known bicyclic nucleoside analogues, that R³ and R⁵ do not togetherdesignate a biradical selected from —CH₂—CH₂—, —O—CH₂—, and—O—Si(^(i)Pr)₂—O—Si(^(i)Pr)₂—O—.

[0169] The monomeric LNAs also comprise basic salts and acid additionsalts thereof. Furthermore, it should be understood that any chemicalgroup (including any nucleobase), which is reactive under the conditionsprevailing in chemical oligonucleotide synthesis, is optionallyfunctional group protected as known in the art. This means that groupssuch as hydroxy, amino, carboxy, sulphono, and mercapto groups, as wellas nucleobases, of a monomeric LNA are optionally functional groupprotected. Protection (and deprotection) is performed by methods knownto the person skilled in the art (see, e.g., Greene, T. W. and Wuts, P.G. M., “Protective Groups in Organic Synthesis”, 2^(nd) ed., John Wiley,N.Y. (1991), and M. J. Gait, Oligonucleotide Synthesis, IRL Press,1984).

[0170] Illustrative examples of hydroxy protection groups are optionallysubstituted trityl, such as 4,4′-dimethoxytrityl (DMT),4-monomethoxytrityl (MMT), and trityl, optionally substituted9-(9-phenyl)xanthenyl (pixyl), optionally substituted ethoxycarbonyloxy,p-phenylazophenyloxycarbonyloxy, tetraahydropyranyl (thp),9-fluorenylmethoxycarbonyl (Fmoc), methoxytetrahydropyranyl (mthp),silyloxy such as trimethylsilyl (TMS), triisopropylsilyl (TIPS),tert-butyldimethylsilyl (TBDMS), triethylsilyl, and phenyldimethylsilyl,benzyloxycarbonyl or substituted benzyloxycarbonyl ethers such as2-bromo benzyloxycarbonyl, tert-butylethers, alkyl ethers such as methylether, acetals (including two hydroxy groups), acyloxy such as acetyl orhalogen substituted acetyls, e.g. chloroacetyl or fluoroacetyl,isobutyryl, pivaloyl, benzoyl and substituted benzoyls, methoxymethyl(MOM), benzyl ethers or substituted benzyl ethers such as2,6-dichlorobenzyl (2,6-Cl₂Bzl). Alternatively, the hydroxy group may beprotected by attachment to a solid support optionally through a linker.

[0171] Illustrative examples of amino protection groups are Fmoc(fluorenylmethoxycarbonyl), BOC (tert-butyloxycarbonyl),trifluoroacetyl, allyloxycarbonyl (alloc, AOC), benzyloxycarbonyl (Z,Cbz), substituted benzyloxycarbonyls such as 2-chloro benzyloxycarbonyl((2-CIZ), monomethoxytrityl (MMT), dimethoxytrityl (DMT), phthaloyl, and9-(9-phenyl)xanthenyl (pixyl).

[0172] Illustrative examples of carboxy protection groups are allylesters, methyl esters, ethyl esters, 2-cyanoethylesters,trimethylsilylethylesters, benzyl esters (Obzl), 2-adamantyl esters(O-2-Ada), cyclohexyl esters (OcHex), 1,3-oxazolines, oxazoler,1,3-oxazolidines, amides or hydrazides.

[0173] Illustrative examples of mercapto protecting groups are trityl(Trt), acetamidomethyl (acm), trimethylacetamidomethyl (Tacm),2,4,6-trimethoxybenzyl (Tmob), tert-butylsulfenyl (StBu),9-fluorenylmethyl (Fm), 3-nitro-2-pyridinesulfenyl (Npys), and4-methylbenzyl (Meb).

[0174] Furthermore, it may be necessary or desirable to protect anynucleobase included in an monomeric LNA, especially when the monomericLNA is to be incorporated in an oligomer according to the invention. Inthe present context, the term “protected nucleobases” means that thenucleobase in question is carrying a protection group selected among thegroups which are well-known for a man skilled in the art (see e.g.Protocols for Oligonucleotides and Analogs, vol 20, (Sudhir Agrawal,ed.), Humana Press, 1993, Totowa, N.J.; S. L. Beaucage and R. P. Iyer,Tetrahedron, 1993, 49, 6123; S. L. Beaucage and R. P. Iyer, Tetrahedron,1992, 48, 2223; and E. Uhlmann and A. Peyman, Chem. Rev., 90, 543.).Illustrative examples are benzoyl, isobutyryl, tert-butyl,tert-butyloxycarbonyl, 4-chloro-benzyloxycarbonyl, 9-fluorenylmethyl,9-fluorenylmethyloxycarbonyl, 4-methoxybenzoyl, 4-methoxytriphenylmethyl, optionally substituted triazolo, p-toluenesulphonyl,optionally substituted sulphonyl, isopropyl, optionally substitutedamidines, optionally substituted trityl, phenoxyacetyl, optionallysubstituted acyl, pixyl, tetrahydropyranyl, optionally substituted silylethers, and 4-methoxybenzyloxycarbonyl. Chapter 1 in “Protocols foroligonucleotide conjugates”, Methods in Molecular Biology, vol 26,(Sudhir Agrawal, ed.), Humana Press, 1993, Totowa, N.J. and S. L.Beaucage and R. P. Iyer, Tetrahedron, 1992, 48, 2223 disclose furthersuitable examples.

[0175] In a preferred embodiment, the group B in a monomeric LNA ispreferably selected from nucleobases and protected nucleobases.

[0176] In an embodiment of the monomeric LNAs according to the presentinvention, one of Q and Q*, preferably Q*, designates a group selectedfrom Act-O—, Act-S—, Act-N(R^(H))—, Act-O—CH₂—, Act-S—CH₂—,Act-N(R^(H))—CH₂—, and the other of Q and Q*, preferably Q, designates agroup selected from hydrogen, azido, halogen, cyano, nitro, hydroxy,Prot-O—, mercapto, Prot-S—, C₁₋₆-alkylthio, amino, Prot-N(R^(H))—, mono-or di(C₁₋₆-alkyl)amino, optionally substituted C₁₋₆-alkoxy, optionallysubstituted C₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl, optionallysubstituted C₂₋₆-alkenyloxy, optionally substituted C₂₋₈-alkynyl,optionally substituted C₂₋₆-alkynyloxy, monophosphate, diphosphate,triphosphate, DNA intercalators, photochemically active groups,thermochemically active groups, chelating groups, reporter groups,ligands, carboxy, sulphono, hydroxymethyl, Prot-O—CH₂—, aminomethyl,Prot-N(R^(H))—CH₂—, carboxymethyl, sulphonomethyl, and R^(H) is selectedfrom hydrogen and C₁₋₆-alkyl.

[0177] In the case described above, the group Prot designates aprotecting group for —OH, —SH, and —NH(R^(H)), respectively. Suchprotection groups are selected from the same as defined above forhydroxy protection groups, mercapto protection group, and aminoprotection groups, respectively, however taking into consideration theneed for a stable and reversible protection group. However, it ispreferred that any protection group for —OH is selected from optionallysubstituted trityl, such as dimethoxytrityl (DMT), monomethoxytrityl(MMT), and trityl, and 9-(9-phenyl)xanthenyl (pixyl), optionallysubstituted, tetrahydropyranyl (thp) (further suitable hydroxyprotection groups for phosphoramidite oligonucleotide synthesis aredescribed in Agrawal, ed. “Protocols for Oligonucleotide Conjugates”;Methods in Molecular Biology, vol. 26, Humana Press, Totowa, N.J. (1994)and Protocols for Oligonucleotides and Analogs, vol 20, (Sudhir Agrawal,ed.), Humana Press, 1993, Totowa, N.J.), or protected as acetal; thatany protection group for —SH is selected from trityl, such asdimethoxytrityl (DMT), monomethoxytrityl (MMT), and trityl, and9-(9-phenyl)xanthenyl (pixyl), optionally substituted, tetrahydropyranyl(thp) (further suitable mercapto protection groups for phosphoramiditeoligonucleotide synthesis are also described in Agrawal (see above); andthat any protecting group for —NH(R^(H)) is selected from trityl, suchas dimethoxytrityl (DMT), monomethoxytrityl (MMT), and trityl, and9-(9-phenyl)xanthenyl (pixyl), optionally substituted, tetrahydropyranyl(thp) (further suitable amino protection groups for phosphoramiditeoligonucleotide synthesis are also described in Agrawal (see above).

[0178] In the embodiment above, as well as for any monomeric LNAsdefined herein, Act designates an activation group for —OH, —SH, and—NH(R^(H)), respectively. Such activation groups are, e.g., selectedfrom optionally substituted O-phosphoramidite, optionally substitutedO-phosphortriester, optionally substituted O-phosphordiester, optionallysubstituted H-phosphonate, and optionally substituted O-phosphonate.

[0179] In the present context, the term “phosphoramidite” means a groupof the formula —P(OR^(x))—N(R^(y))₂, wherein R^(x) designates anoptionally substituted alkyl group, e.g. methyl, 2-cyanoethyl, orbenzyl, and each of R^(y) designate optionally substituted alkyl groups,e.g. ethyl or isopropyl, or the group —N(R^(y))₂ forms a morpholinogroup (—N(CH₂CH₂)₂O). R^(x) preferably designates 2-cyanoethyl and thetwo ^(Ry) are preferably identical and designate isopropyl. Thus, anespecially relevant phosphoramidite isN,N-diisopropyl-O-(2-cyanoethyl)phosphoramidite.

[0180] It should be understood that the protecting groups used hereinfor a single, monomeric LNA or several monomeric LNAs may be selected sothat when this/these LNA(s) are incorporated in an oligomer according tothe invention, it will be possible to perform either a simultaneousdeprotection or a sequential deprotection of the functional groups. Thelatter situation opens for the possibility of regioselectivelyintroducing one or several “active/functional” groups such as DNAintercalators, photochemically active groups, thermochemically activegroups, chelating groups, reporter groups, and ligands, where suchgroups may be attached via a spacer as described above.

[0181] In a preferred embodiment, Q is selected from hydrogen, azido,halogen, cyano, nitro, hydroxy, Prot-O—, mercapto, Prot-S—,C₁₋₆-alkylthio, amino, Prot-N(R^(H))—, mono- or di(C₁₋₆-alkyl)amino,optionally substituted C₁₋₆-alkoxy, optionally substituted C₁₋₆-alkyl,optionally substituted C₂₋₆-alkenyl, optionally substitutedC₂₋₆-alkenyloxy, optionally substituted C₂₋₆-alkynyl, optionallysubstituted C₂₋₆-alkynyloxy, monophosphate, diphosphate, triphosphate,DNA intercalators, photochemically active groups, thermochemicallyactive groups, chelating groups, reporter groups, ligands, carboxy,sulphono, hydroxymethyl, Prot-O—CH₂—, aminomethyl, Prot-N(R^(H))—CH₂—,carboxymethyl, sulphonomethyl, where Prot is a protection group for —OH,—SH, and —NH(R^(H)), respectively, and R^(H) is selected from hydrogenand C₁₋₆-alkyl; and Q is selected from hydrogen, azido, halogen, cyano,nitro, hydroxy, Act-O—, mercapto, Act-S—, C₁₋₆-alkylthio, amino,Act-N(R^(H))—, mono- or di(C₁₋₆-alkyl)amino, optionally substituted C₁—,alkoxy, optionally substituted C₁₋₆-alkyl, optionally substitutedC₂₋₆-alkenyl, optionally substituted C₂₋₆-alkenyloxy, optionallysubstituted C₂₋₆-alkynyl, optionally substituted C₂₋₆-alkynyloxy, DNAintercalators, photochemically active groups, thermochemically activegroups, chelating groups, reporter groups, ligands, carboxy, sulphono,where Act is an activation group for —OH, —SH, and —NH(R^(H)),respectively, and R^(H) is selected from hydrogen and C₁₋₆-alkyl.

[0182] The monomeric LNAs of the general formula II may, as the LNAsincorporated into oligomers, represent various stereoisomers. Thus, thestereochemical variants described above for the LNAs incorporated intooligomers are believed to be equally applicable in the case of monomericLNAs (however, it should be noted that P should then be replaced withQ).

[0183] In a preferred embodiment of the present invention, the monomericLNA has the general formula IIa

[0184] wherein the substituents are defined as above.

[0185] Furthermore, with respect to the definitions of substituents,biradicals, R., etc. the same preferred embodiments as defined above forthe oligomer according to the invention also apply in the case ofmonomeric LNAs.

[0186] In a particularly interesting embodiment of the monomeric LNAs ofthe present invention, B designates a nucleobase, preferably anucleobase selected from thymine, cytosine, urasil, adenine and guanine(in particular adenine and guanine), X is —O—, R^(2*) and R^(4*)together designate a biradical selected from —(CH₂)₀₋₁—O—(CH₂)₁₋₃—,—(CH₂)₀₋₁—S—(CH₂)₁₋₃—, and —(CH₂)₀₋₁—N(R^(N))—(CH₂)₁₋₃—, in particular—O—CH₂—, —S—CH₂— and —R^(N)—CH₂—, where R^(N) is selected from hydrogenand C₁₋₄-alkyl, Q designates Prot-O—, R^(3*) is Q* which designatesAct-OH, and R^(1*), R², R³, R⁵, and R^(5*) each designate hydrogen. Inthis embodiment, R^(N) may also be selected from DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups and ligands.

[0187] In a further particularly interesting embodiment of the monomericLNAs of the present invention, B designates a nucleobase, preferably anucleobase selected from thymine, cytosine, urasil, adenine and guanine(in particular adenine and guanine), X is —O—, R^(2*) and R^(4*)together designate a biradical selected from —(CH₂)₀₋₁—O—(CH₂)₁₋₃—,—(CH₂)₀₋₁—S—(CH₂)₁₋₃—, and —(CH₂)₀₋₁—N(R^(N))—(CH₂)₁₋₃—, in particular—O—CH₂—, —S—CH₂— and —R^(N)—CH₂—, where R^(N) is selected from hydrogenand C₁₋₄-alkyl, Q is selected from hydroxy, mercapto, C₁₋₆-alkylthio,amino, mono- or di(C₁₋₆-alkyl)amino, optionally substituted C₁₋₆-alkoxy,optionally substituted C₂₋₆-alkenyloxy, optionally substitutedC₂₋₆-alkynyloxy, monophosphate, diphosphate, and triphosphate, R^(3*) isQ* which is selected from hydrogen, azido, halogen, cyano, nitro,hydroxy, mercapto, C₁₋₆-alkylthio, amino, mono- or di(C₁₋₆-alkyl)amino,optionally substituted C₁₋₆-alkoxy, optionally substituted C₁₋₆-alkyl,optionally substituted C₂₋₆-alkenyl, optionally substitutedC₂₋₆-alkenyloxy, optionally substituted C₂₋₆-alkynyl, and optionallysubstituted C₂₋₆-alkynyloxy, R³ is selected from hydrogen, optionallysubstituted C₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl, andoptionally substituted C₂₋₆-alkynyl, and R^(1*), R², R⁵, and R^(5*) eachdesignate hydrogen. Also here, R^(N) may also be selected from DNAintercalators, photochemically active groups, thermochemically activegroups, chelating groups, reporter groups and ligands.

[0188] In a further particularly interesting embodiment of the monomericLNAs of the present invention, B designates a nucleobase, X is —O—, R²and R³ together designate a biradical selected from —(CH₂)₀₋₁—O—CH═CH—,—(CH₂)₀₋₁—S—CH═CH—, and —(CH₂)₀₋₁—N(R^(N))—CH═CH— where R^(N) isselected from hydrogen and C₁₋₄-alkyl, Q is selected from hydroxy,mercapto, C₁₋₆-alkylthio, amino, mono- or di(C₁₋₆-alkyl)amino,optionally substituted C₁₋₆-alkoxy, optionally substitutedC₂₋₆-alkenyloxy, optionally substituted C₂₋₆-alkynyloxy, monophosphate,diphosphate, and triphosphate, R^(3*) is Q* which is selected fromhydrogen, azido, halogen, cyano, nitro, hydroxy, mercapto,C₁₋₆-alkylthio, amino, mono- or di(C₁₋₆-alkyl)amino, optionallysubstituted C₁₋₆-alkoxy, optionally substituted C₁₋₆-alkyl, optionallysubstituted C₂₋₆-alkenyl, optionally substituted C₂₋₆-alkenyloxy,optionally substituted C₂₋₆-alkynyl, and optionally substitutedC₂₋₆-alkynyloxy, and R^(1*), R^(2*), R^(4*), R⁵, and R^(5*) eachdesignate hydrogen.

[0189] One aspect of the invention is to provide various derivatives ofLNAs for solid-phase and/or solution phase incorporation into anoligomer. As an illustrative example, monomers suitable forincorporation of(1S,3R,4R,7S)-7-hydroxy-1-hydroxymethyl-3-(thymin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane,(1S,3R,4R,7S)-7-hydroxy-1-hydroxymethyl-3-(cytosin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane,(1S,3R,4R,7S)-7-hydroxy-1-hydroxymethyl-3-(urasil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane,(1S,3R,4R,7S)-7-hydroxy-1-hydroxymethyl-3-(guanin-1-yl)-2,5-dioxabicyclo-[2.2.1]heptane,and(1S,3R,4R,7S)-7-hydroxy-1-hydroxymethyl-3-(adenin-1-yl)-2,5-dioxabicyclo[2.2.1]heptaneusing the phosphoramidite approach, the phosphortriester approach, andthe H-phosphonate approach, respectively, are(1R,3R,4R,7S)-7-(2-Cyanoethoxy(diisopropylamino)phosphinoxy)-1-(4,4′-dimethoxytrityloxymethyl)-3-(thymin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane,(1R,3R,4R,7S)-7-hydroxy-1-(4,4′-dimethoxytrityloxymethyl)-3-(thymin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane-7-O-(2-chlorophenylphosphate),and(1R,3R,4R,7S)-7-hydroxy-1-(4,4′-dimethoxytrityloxymethyl)-3-(thymin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane-7-O—(H-phosphonate)and the 3-(cytosin-1-yl), 3-(urasil-1-yl), 3-(adenin-1-yl) and3-(guanin-1-yl) analogues thereof, respectively. Furthermore, theanalogues where the methyleneoxy biradical of the monomers issubstituted with a methylenethio, a methyleneamino, or a 1,2-ethylenebiradical are also expected to constitute particularly interestingvariants within the present invention. The methylenethio andmethyleneamino analogues are believed to equally applicable as themethyleneoxy analogue and therefore the specific reagents correspondingto those mentioned for incorporation of(1S,3R,4R,7S)-7-hydroxy-1-hydroxymethyl-3-(thymin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane,(1S,3R,4R,7S)-7-hydroxy-1-hydroxymethyl-3-(cytosin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane,(1S,3R,4R,7S)-7-hydroxy-1-hydroxymethyl-3-(urasil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane,(1S,3R,4R,7S)-7-hydroxy-1-hydroxymethyl-3-(guanin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane,and(1S,3R,4R,7S)-7-hydroxy-1-hydroxymethyl-3-(adenin-1-yl)-2,5-dioxabicyclo[2.2.1]heptaneshould also be considered as particularly interesting reactive monomerswithin the present invention. For the methyleneamine analogue, it shouldbe noted that the secondary amine may carry a substituent selected fromoptionally substituted C₁₋₆-alkyl such as methyl and benzyl, optionallysubstituted C₁₋₆-alkylcarbonyl such as trifluoroacetyl, optionallysubstituted arylcarbonyl and optionally substituted heteroarylcarbonyl.

[0190] In a particularly interesting embodiment, the present inventionrelates to an oligomer comprising at least one LNA of the generalformula Ia

[0191] wherein X is —O—; B is selected from nucleobases, DNAintercalators, photochemically active groups, thermochemically activegroups, chelating groups, reporter groups, and ligands; P designates theradical position for an internucleoside linkage to a succeeding monomer,or a 5′-terminal group, such internucleoside linkage or 5′-terminalgroup optionally including the substituent R⁵; R^(3*) is a group P*which designates an internucleoside linkage to a preceding monomer, or a3′-terminal group; R² and R^(4*) together designate a biradical selectedfrom —O—, —S, —N(R*)—, —(CR*R*)_(r+s+1)—, (CR*R*)_(r)—O—(CR*R*)_(s)—,—(CR*R*)_(r)—S—(CR*R*)_(s)—, —(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—,—O—(CR*R*)_(r+s)—O—, —S—(CR*R*)_(r+s)—O—, —O—(CR*R*)_(r+s)—S—,—N(R*)—(CR*R*)_(r+s)—O—, —O—(CR*R*)_(r+s)—N(R*)—, —S—(CR*R*)_(r+s)—S—,—N(R*)—(CR*R*)_(r+s)—N(R*)—, —N(R*)—(CR*R*)_(r+s)—S—, and—S—(CR*R*)_(r+s)—N(R*)—; wherein each R* is independently selected fromhydrogen, halogen, azido, cyano, nitro, hydroxy, mercapto, amino, mono-or di(C₁₋₆-alkyl)amino, optionally substituted C₁₋₆-alkoxy, optionallysubstituted C₁₋₆-alkyl, DNA intercalators, photochemically activegroups, thermochemically active groups, chelating groups, reportergroups, and ligands, and/or two adjacent (non-geminal) R* may togetherdesignate a double bond, and each of r and s is 0-3 with the provisothat the sum r+s is 1-4; each of the substituents R^(1*), R², R³, R⁵,and R^(5*) is independently selected from hydrogen, optionallysubstituted C₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl, hydroxy,C₁₋₆-alkoxy, C₂₋₆-alkenyloxy, carboxy, C₁₋₆-alkoxycarbonyl,C₁₋₆-alkylcarbonyl, formyl, amino, mono- and di(C₁₋₆-alkyl)amino,carbamoyl, mono- and di(C₁₋₆-alkyl)-amino-carbonyl,C₁₋₆-alkylcarbonylamino, carbamido, azido, C₁₋₆-alkanoyloxy, sulphono,sulphanyl, C₁₋₆-alkylthio, DNA intercalators, photochemically activegroups, thermochemically active groups, chelating groups, reportergroups, and ligands, and halogen, where two geminal substituentstogether may designate oxo; and basic salts and acid addition saltsthereof. In particular, one R* is selected from hydrogen, hydroxy,optionally substituted C₁₋₆-alkoxy, optionally substituted C₁₋₆-alkyl,DNA intercalators, photochemically active groups, thermochemicallyactive groups, chelating groups, reporter groups, and ligands, and anyremaining substituents R are hydrogen. Especially, the biradical isselected from —O—, —(CH₂)₀₋₁—O—(CH₂)₁₋₃—, —(CH₂)₀₋₁—S—(CH₂)₁₋₃—,—(CH₂)₀₋₁—N(R^(N))—(CH₂)₁₋₃—, and —(CH₂)₂₋₄—.

[0192] In a further particularly interesting embodiment, the presentinvention relates to an LNA of the general formula IIa

[0193] wherein X is —O—; B is selected from nucleobases, DNAintercalators, photochemically active groups, thermochemically activegroups, chelating groups, reporter groups, and ligands; R^(3*) is agroup Q*; each of Q and Q* is independently selected from hydrogen,azido, halogen, cyano, nitro, hydroxy, Prot-O—, Act-O—, mercapto,Prot-S—, Act-S—, C₁₋₆-alkylthio, amino, Prot-N(R^(H))—, Act-N(R^(H))—,mono- or di(C₁₋₆-alkyl)amino, optionally substituted C₁₋₆-alkoxy,optionally substituted C₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl,optionally substituted C₂₋₆-alkenyloxy, optionally substitutedC₂₋₆-alkynyl, optionally substituted C₂₋₆-alkynyloxy, monophosphate,diphosphate, triphosphate, DNA intercalators, photochemically activegroups, thermochemically active groups, chelating groups, reportergroups, ligands, carboxy, sulphono, hydroxymethyl, Prot-O—CH₂—,Act-O—CH₂—, aminomethyl, Prot-N(R^(H))—CH₂—, Act-N(R^(H))—CH₂—,carboxymethyl, sulphonomethyl, where Prot is a protection group for —OH,—SH, and —NH(R^(H)), respectively, Act is an activation group for —OH,—SH, and —NH(R^(H)), respectively, and R^(H) is selected from hydrogenand C₁₋₆-alkyl; R^(2*) and R^(4*) together designate a biradicalselected from —O—, —S, —N(R*)—, —(CR*R*)_(r+s+1)—,(CR*R*)_(r)—O—(CR*R*)_(s)—, —(CR*R*)_(r)—S—(CR*R*)_(s)—,—(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—, —O—(CR*R*)_(r+s)—O—,—S—(CR*R*)_(r+s)—O—, —O—(CR*R*)_(r+s)—S—, —N(R*)—(CR*R*)_(r+s)—O—,—O—(CR*R*)_(r+s)—N(R*)—, —S—(CR*R*)_(r+s)—S—,—N(R*)—(CR*R*)_(r+s)—N(R*)—, —N(R*)—(CR*R*)_(r+s)—S—, and—S—(CR*R*)_(r+s)—N(R*)—; wherein each R* is independently selected fromhydrogen, halogen, azido, cyano, nitro, hydroxy, mercapto, amino, mono-or di(C₁₋₆-alkyl)amino, optionally substituted C₁₋₆-alkoxy, optionallysubstituted C₁₋₆-alkyl, DNA intercalators, photochemically activegroups, thermochemically active groups, chelating groups, reportergroups, and ligands, and/or two adjacent (non-geminal) R* may togetherdesignate a double bond, and each of r and s is 0-3 with the provisothat the sum r+s is 1-4; each of the substituents R^(1*), R², R³, R⁵,and R^(5*) is independently selected from hydrogen, optionallysubstituted C₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl, hydroxy,C₁₋₆-alkoxy, C₂₋₆-alkenyloxy, carboxy, C₁₋₆-alkoxycarbonyl,C₁₋₆alkylcarbonyl, formyl, amino, mono- and di(C₁₋₆-alkyl)amino,carbamoyl, mono- and di(C₁₋₆-alkyl)-amino-carbonyl,C₁₋₆-alkyl-carbonylamino, carbamido, azido, C₁₋₆-alkanoyloxy, sulphono,sulphanyl, C₁₋₆-alkylthio, DNA intercalators, photochemically activegroups, thermochemically active groups, chelating groups, reportergroups, and ligands, and halogen, where two geminal substituentstogether may designate oxo; and basic salts and acid addition saltsthereof; and with the proviso that any chemical group (including anynucleobase), which is reactive under the conditions prevailing inoligonucleotide synthesis, is optionally functional group protected.Preferably, one R is selected from hydrogen, hydroxy, optionallysubstituted C₁₋₆-alkoxy, optionally substituted C₁₋₆-alkyl, DNAintercalators, photochemically active groups, thermochemically activegroups, chelating groups, reporter groups, and ligands, and anyremaining substituents R* are hydrogen. Especially, the biradical isselected from —O—, —(CH₂)₀₋₁—O—(CH₂)₁₋₃—, —(CH₂)₀₋₁—S—(CH₂)₁₋₃—,—(CH₂)₀₋₁—N(R^(N))—(CH₂)₁₋₃—, and —(CH₂)₂₋₄—.

[0194] Generally, the present invention provides oligomers havingsurprisingly good hybridisation properties with respect to affinity andspecificity. Thus, the present invention provides an oligomer comprisingat least one nucleoside analogue which imparts to the oligomer a T_(m)with a complementary DNA oligonucleotide which is at least 2.5° C.higher, preferably at least 3.5° C. higher, in particular at least 4.0°C. higher, especially at least 5.0° C. higher, than that of thecorresponding unmodified reference oligonucleotide which does notcomprise any nucleoside analogue. In particular, the T_(m) of theoligomer is at least 2.5×N° C. higher, preferably at least 3.5×N° C.higher, in particular at least 4.0×N° C. higher, especially at least5.0×N° C. higher, where N is the number of nucleoside analogues.

[0195] In the case of hybridisation with a complementary RNAoligonucleotide, the at least one nucleoside analogue imparts to theoligomer a T_(m) with the complementary DNA oligonucleotide which is atleast 4.0° C. higher, preferably at least 5.0° C. higher, in particularat least 6.0° C. higher, especially at least 7.0° C. higher, than thatof the corresponding unmodified reference oligonucleotide which does notcomprise any nucleoside analogue. In particular, the T_(m) of theoligomer is at least 4.0×N° C. higher, preferably at least 5.0×N° C.higher, in particular at least 6.0×N° C. higher, especially at least7.0×N° C. higher, where N is the number of nucleoside analogues.

[0196] The term “corresponding unmodified reference oligonucleotide” isintended to mean an oligonucleotide solely consisting of naturallyoccurring nucleotides which represents the same nucleobases in the sameabsolute order (and the same orientation).

[0197] The T_(m) is measured under one of the following conditions (i.e.essentially as illustrated in Example 129):

[0198] a) 10 mM Na₂HPO₄, pH 7.0, 100 mM NaCl, 0.1 mM EDTA;

[0199] b) 10 mM Na₂HPO₄ pH 7.0, 0.1 mM EDTA; or

[0200] c) 3M tetrametylammoniumchlorid (TMAC), 10 mM Na₂HPO₄, pH 7.0,0.1 mM EDTA;

[0201] preferably under conditions a), at equimolar amounts (typically1.0 μM) of the oligomer and the complementary DNA oligonucleotide.

[0202] The oligomer is preferably as defined above, where the at leastone nucleoside analogue has the formula I where B is a nucleobase. Inparticular interesting is the cases where at least one nucleosideanalogue includes a nucleobase selected from adenine and guanine.

[0203] Furthermore, with respect to specificity and affinity, theoligomer, when hybridised with a partially complementary DNAoligonucleotide, or a partially complementary RNA oligonucleotide,having one or more mismatches with said oligomer, should exhibit areduction in T_(m), as a result of said mismatches, which is equal to orgreater than the reduction which would be observed with thecorresponding unmodified reference oligonucleotide which does notcomprise any nucleoside analogues. Also, the oligomer should havesubstantially the same sensitivity of T_(m) to the ionic strength of thehybridisation buffer as that of the corresponding unmodified referenceoligonucleotide.

[0204] Oligomers defined herein are typically at least 30% modified,preferably at least 50% modified, in particular 70% modified, and insome interesting applications 100% modified.

[0205] The oligomers of the invention has substantially higher3′-exonucleolytic stability than the corresponding unmodified referenceoligonucleotide. This important property can be examined as described inExample 136.

[0206] Definitions

[0207] In the present context, the term “C₁₋₁₂-alkyl” means a linear,cyclic or branched hydrocarbon group having 1 to 12 carbon atoms, suchas methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, tert-butyl,iso-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, anddodecyl. Analogously, the term “C₁₋₆-alkyl” means a linear, cyclic orbranched hydrocarbon group having 1 to 6 carbon atoms, such as methyl,ethyl, propyl, iso-propyl, pentyl, cyclopentyl, hexyl, cyclohexyl, andthe term “C₁₋₄-alkyl” is intended to cover linear, cyclic or branchedhydrocarbon groups having 1 to 4 carbon atoms, e.g. methyl, ethyl,propyl, iso-propyl, cyclopropyl, butyl, iso-butyl, tert-butyl,cyclobutyl.

[0208] Preferred examples of “C₁₋₆-alkyl” are methyl, ethyl, propyl,iso-propyl, butyl, tert-butyl, iso-butyl, pentyl, cyclopentyl, hexyl,cyclohexyl, in particular methyl, ethyl, propyl, iso-propyl, tert-butyl,iso-butyl and cyclohexyl. Preferred examples of “C₁₋₄-alkyl” are methyl,ethyl, propyl, iso-propyl, butyl, tert-butyl, and iso-butyl.

[0209] Similarly, the term “C₂₋₁₂-alkenyl” covers linear, cyclic orbranched hydrocarbon groups having 2 to 12 carbon atoms and comprisingone unsaturated bond. Examples of alkenyl groups are vinyl, allyl,butenyl, pentenyl, hexenyl, heptenyl, octenyl, dodecaenyl. Analogously,the term “C₂₋₆-alkenyl” is intended to cover linear, cyclic or branchedhydrocarbon groups having 2 to 6 carbon atoms and comprising oneunsaturated bond. Preferred examples of alkenyl are vinyl, allyl,butenyl, especially allyl.

[0210] Similarly, the term “C₂₋₁₂-alkynyl” means a linear or branchedhydrocarbon group having 2 to 12 carbon atoms and comprising a triplebond. Examples hereof are ethynyl, propynyl, butynyl, octynyl, anddodecanyl.

[0211] In the present context, i.e. in connection with the terms“alkyl”, “alkenyl”, and “alkynyl”, the term “optionally substituted”means that the group in question may be substituted one or severaltimes, preferably 1-3 times, with group(s) selected from hydroxy (whichwhen bound to an unsaturated carbon atom may be present in thetautomeric keto form), C₁₋₆-alkoxy (i.e. C₁₋₆-alkyl-oxy),C₂₋₆-alkenyloxy, carboxy, oxo (forming a keto or aldehydefunctionality), C₁₋₆-alkoxycarbonyl, C₁₋₆-alkylcarbonyl, formyl, aryl,aryloxycarbonyl, aryloxy, arylcarbonyl, heteroaryl,heteroaryloxycarbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di(C₁₋₆-alkyl)amino; carbamoyl, mono- anddi(C₁₋₆-alkyl)aminocarbonyl, amino-C₁₋₆-alkyl-aminocarbonyl, mono- anddi(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl, C₁₋₆-alkylcarbonylamino,cyano, guanidino, carbamido, C₁₋₆-alkanoyloxy, sulphono,C₁₋₆-alkylsulphonyloxy, nitro, sulphanyl, C₁₋₆-alkylthio, halogen, whereany aryl and heteroaryl may be substituted as specifically describebelow for “optionally substituted aryl and heteroaryl”.

[0212] Preferably, the substituents are selected from hydroxy,C₁₋₆-alkoxy, carboxy, C₁₋₆-alkoxycarbonyl, C₁₋₆-alkylcarbonyl, formyl,aryl, aryloxycarbonyl, arylcarbonyl, heteroaryl, amino, mono- anddi(C₁₋₆-alkyl)amino, carbamoyl, mono- and di(C₁₋₆-alkyl)aminocarbonyl,amino-C₁₋₆-alkyl-aminocarbonyl, mono- anddi(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl, C₁₋₆-alkylcarbonylamino,cyano, carbamido, halogen, where aryl and heteroaryl may be substituted1-5 times, preferably 1-3 times, with C₁₋₄-alkyl, C₁₋₄-alkoxy, nitro,cyano, amino or halogen. Especially preferred examples are hydroxy,C₁₋₆-alkoxy, carboxy, aryl, heteroaryl, amino, mono- anddi(C₁₋₆-alkyl)amino, and halogen, where aryl and heteroaryl may besubstituted 1-3 times with C₁₋₄-alkyl, C₁₋₄-alkoxy, nitro, cyano, aminoor halogen.

[0213] In the present context the term “aryl” means a fully or partiallyaromatic carbocyclic ring or ring system, such as phenyl, naphthyl,1,2,3,4-tetrahydronaphthyl, anthracyl, phenanthracyl, pyrenyl,benzopyrenyl, fluorenyl and xanthenyl, among which phenyl is a preferredexample.

[0214] The term “heteroaryl” means a fully or partially aromaticcarbocyclic ring or ring system where one or more of the carbon atomshave been replaced with heteroatoms, e.g. nitrogen (═N— or —NH),sulphur, and/or oxygen atoms. Examples of such heteroaryl groups areoxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrrolyl, imidazolyl,pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl, piperidinyl, coumaryl,furyl, quinolyl, benzothiazolyl, benzotriazolyl, benzodiazolyl,benzooxozolyl, phthalazinyl, phthalanyl, triazolyl, tetrazolyl,isoquinolyl, acridinyl, carbazolyl, dibenzazepinyl, indolyl,benzopyrazolyl, phenoxazonyl.

[0215] In the present context, i.e. in connection with the terms “aryl”and “heteroaryl”, the term “optionally substituted” means that the groupin question may be substituted one or several times, preferably 1-5times, in particular 1-3 times) with group(s) selected from hydroxy(which when present in an enol system may be represented in thetautomeric keto form), C₁₋₆-alkyl, C₁₋₆-alkoxy, oxo (which may berepresented in the tautomeric enol form), carboxy, C₁₋₆-alkoxycarbonyl,C₁₋₆-alkylcarbonyl, formyl, aryl, aryloxy, aryloxycarbonyl,arylcarbonyl, heteroaryl, amino, mono- and di(C₁₋₆-alkyl)amino;carbamoyl, mono- and di(C₁₋₆-alkyl)aminocarbonyl,amino-C₁₋₆-alkylaminocarbonyl, mono- anddi(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl, C₁₋₆-alkylcarbonylamino,cyano, guanidino, carbamido, C₁₋₆-alkanoyloxy, sulphono,C₁₋₆-alkylsulphonyloxy, nitro, sulphanyl, dihalogen-C₁₋₄-alkyl,trihalogen-C₁₋₄-alkyl, halogen, where aryl and heteroaryl representingsubstituents may be substituted 1-3 times with C₁₋₄-alkyl, C₁₋₄-alkoxy,nitro, cyano, amino or halogen. Preferred examples are hydroxy,C₁₋₆-alkyl, C₁₋₆-alkoxy, carboxy, C₁₋₆-alkoxycarbonyl,C₁₋₆-alkylcarbonyl, aryl, amino, mono- and di(C₁₋₆-alkyl)amino, andhalogen, wherein aryl may be substituted 1-3 times with C₁₋₄-alkyl,C₁₋₄-alkoxy, nitro, cyano, amino or halogen.

[0216] “Halogen” includes fluoro, chloro, bromo, and iodo.

[0217] It should be understood that oligomers (wherein LNAs areincorporated) and LNAs as such include possible salts thereof, of whichpharmaceutically acceptable salts are especially relevant. Salts includeacid addition salts and basic salts. Examples of acid addition salts arehydrochloride salts, sodium salts, calcium salts, potassium salts, etc.Examples of basic salts are salts where the (remaining) counter ion isselected from alkali metals, such as sodium and potassium, alkalineearth metals, such as calcium, and ammonium ions (+N(R^(g))₃R^(h), whereeach of R^(g) and R^(h) independently designates optionally substitutedC₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl, optionally substitutedaryl, or optionally substituted heteroaryl). Pharmaceutically acceptablesalts are, e.g., those described in Remington's Pharmaceutical Sciences,17. Ed. Alfonso R. Gennaro (Ed.), Mack Publishing Company, Easton, Pa.,U.S.A., 1985 and more recent editions and in Encyclopedia ofPharmaceutical Technology. Thus, the term “an acid addition salt or abasic salt thereof” used herein is intended to comprise such salts.Furthermore, the oligomers and LNAs as well as any intermediates orstarting materials therefor may also be present in hydrate form.

[0218] Preparation of Monomers

[0219] In a preferred embodiment, nucleosides containing an additional2′-O,4′-C-linked ring were synthesised by the following procedure:

[0220] Synthesis of a number of 4′-C-hydroxymethyl nucleosides have beenreported earlier (R. D. Youssefyeh, J. P. H. Verheyden and J. G.Moffatt, J. Org. Chem., 1979, 44, 1301; G. H. Jones, M. Taniguchi, D.Tegg and J. G. Moffatt, J. Org. Chem., 1979, 44, 1309; C. O-Yang, H. Y.Wu, E. B. Fraser-Smith and K. A. M. Walker, Tetrahedron Lett., 1992, 33,37; H. Thrane, J. Fensholdt, M. Regner and J. Wengel, Tetrahedron, 1995,51, 10389; K. D. Nielsen, F. Kirpekar, P. Roepstorff and J. Wengel,Bioorg. Med. Chem., 1995, 3, 1493). For exemplification of synthesis of2′-O,4′-C-linked bicyclic nucleosides we chose a strategy starting from4′-C-hydroxymethyl furanose derivative 31. Benzylation, acetylation, andacetolysis followed by another acetylation afforded furanose 33, a keyintermediate for nucleoside coupling. Stereoselective reaction withsilylated thymine afforded compound 34 which was deacetylated to givenucleoside diol 35. Tosylation followed by base-induced ring closureafforded the 2′-O,4′-C-linked bicyclic nucleoside derivative 36.Debenzylation yielded the unprotected bicyclic nucleoside analogue 37which was transformed into the 5′-O-4,4′-dimethoxytrityl protectedanalogue 38 and subsequently into the phosphoramidite derivative 39 foroligonucleotide synthesis. A similar procedure has been used forsynthesis of the corresponding uracil, adenine, cytosine and guaninenucleosides as exemplified in the example section. This coupling methodis only one of several possible as will be apparent for a person skilledin the art. A strategy starting from a preformed nucleoside is alsopossible Thus, for example, conversion of uridine derivative 62 toderivative 44 was successfully accomplished by tosylation,deisopropylidination and base-induced ring-closure. As another example,conversion of nucleoside 67 into nucleoside 61B has been accomplished asdepicted in FIG. 34. Conversion of the bicyclic thymine nucleoside 37into the corresponding 5-methylcytosine nucleoside 65 was accomplishedby a known reaction sequence using triazole and POCl₃ followed bybenzoylation and treatment by ammonia. A similar procedure should beapplicable for the synthesis of 57C from 44. As another example ofpossible strategies, coupling of precyclised furanose derivativesalready containing an additional ring with nucleobase derivatives ispossible. Such a strategy would in addition allow preparation of thecorresponding α-nucleoside analogues. When coupling with a protectedmethyl furanoside of 4-C,2-O-methylene-D-ribofuranose, we obtainedmainly a ring-opened product. However, coupling of 1-O-acetyl furanose207 or thiophenyl furanose 212 yielded successfully LNA nucleosides withthe α-anomers as one product. Incorporation of such (LNA nucleosideswill be possible using the standard oligomerisation techniques (as forthe LNA oligomers containing Z) yielding α-LNA oligomers. In addition, asynthetic strategy performing nucleoside coupling using a4′-C-hydroxymethyl furanose already activated for ring closure (e.g. bycontaining a mesyl or tosyl group at the 4′-C-hydroxymethyl group), ispossible as exemplified by conversion of furanose 78 to nucleoside 79followed by deprotection and ring closure to give 36. Chemical orenzymatic transglycosylation or anomerisation of appropriate furanosederivatives or nucleosides are yet other possible synthetic strategies.These nd other related strategies allow for synthesis of bicyclicnucleosides containing other nucleobases or analogues thereof by eithercoupling with these nucleobases or analogues, or starting from preformednucleoside derivatives.

[0221] The described examples are meant to be illustrative for theprocedures and examples of this invention. The structures of thesynthesised compounds were verified using 1D or 2D NMR techniques, e.g.NOE experiments.

[0222] An additional embodiment of the present invention is to providebicyclic nucleosides containing additional rings of different sizes andof different chemical structures. From the methods described it isobvious for a person skilled in the art of organic synthesis thatcyclisation of other nucleosides is possible using similar procedures,also of nucleosides containing different C-branches. The person skilledin the art will be able to find inspiration and guidance for thepreparation of substituted nucleoside analogue intermediates in theliterature, see e.g. WO 96/14329. Regarding rings of different chemicalcompositions it is clear that using similar procedures or procedureswell-established in the field of organic chemistry, synthesis of forexample thio analogues of the exemplified oxo analogues is possible asis the synthesis of the corresponding amino analogues (using for examplenucleophilic substitution reactions or reductive alkylations).

[0223] In the example section, synthesis of the amino LNA analogues73-74F are described. Conversion of 74 and 74D into standard buildingblocks for oligomerisation was possible by 5′-O-DMT protection and3′-O-phosphitylation following the standard procedures. For the aminoLNA analogue, protection of the 2′-amino functionality is needed forcontrolled linear oligomerisation. Such protection can be accomplishedusing standard amino group protection techniques like, e.g., Fmoc,trifluoroacetyl or BOC. Alternatively, an N-alkyl group (e.g. benzyl,methyl, ethyl, propyl or functionalised alkyl) can be kept on duringnucleoside transformations and oligomerisation. In FIGS. 35 and 36,strategies using N-trifluoroacetyl and N-methyl derivatives are shown.As outlined in FIG. 37, conversion of nucleoside 75 into the 2′-thio-LNAnucleoside analogue 76D has been successfully performed as has thesubsequent syntheses of the phosphoramidite derivative 76F. Compound 76Fhas the required structure for automated synthesis of 2′-thio-LNAoligonucleotides. The N-trifluoroacetyl 2′-amino-LNA synthon 74A has therequired structure for automated synthesis of 2′-amino-LNAoligonucleotides.

[0224] Synthesis of the corresponding cytosine, guanine, and adeninederivatives of the 2′-thio and 2′-amino LNA nucleosides can beaccomplished using strategies analogous to those shown in FIGS. 35, 36and 37. Alternative, the stereochemistry around C-2′ can be invertedbefore cyclisations either by using a conveniently configurated, e.g. anarabino-configurated, furanose synthon, or by inverting theconfiguration around the C-2′ carbon atom starting from aribo-configurated nucleoside/furanose. Subsequent activation of the2′-β-OH, e.g. by tosylation, double nucleophilic substitution as in theurasil/thymine example described above, could furnish the desiredbicyclic 2′-thio-LNA or 2′-amino-LNA nucleosides. The thus obtainedproperly protected cytosine, guanine, and adenine analogues can beprepared for oligomerisation using the standard reactions(DMT-protection and phosphitylation) as described above for otherexamples.

[0225] Preparation of Oligomers

[0226] Linear-, branched-(M. Grotli and B. S. Sproat, J. Chem. Soc.,Chem. Commun., 1995, 495; R. H. E. Hudson and M. J. Damha, J. Am. Chem.Soc., 1993, 115, 2119; M. Von Büren, G. V. Petersen, K. Rasmussen, G.Brandenburg, J. Wengel and F. Kirpekar, Tetrahedron, 1995, 51, 8491) andcircular (G. Prakash and E. T. Kool, J. Am. Chem. Soc., 1992, 114, 3523)Oligo- and polynucleotides of the invention may be produced using thepolymerisation techniques of nucleic acid chemistry well known to aperson of ordinary skill in the art of organic chemistry.Phosphoramidite chemistry (S. L. Beaucage and R. P. Iyer, Tetrahedron,1993, 49, 6123; S. L. Beaucage and R. P. Iyer, Tetrahedron, 1992, 48,2223) was used, but e.g. H-phosphonate chemistry, phosphortriesterchemistry or enzymatic synthesis could also be used. Generally, standardcoupling conditions and the phosphoramidite approach was used, but forsome monomers of the invention longer coupling time, and/or repeatedcouplings with fresh reagents, and/or use of more concentrated couplingreagents were used. As another possibility, activators more active than1H-tetrazole could also be used to increase the rate of the couplingreaction. The phosphoramidietes 39, 46, 53, 57D, 61D, and 66 all coupledwith satisfactory >95% step-wise coupling yields. Anall-phosphorothioate LNA oligomer (Table 7) was synthesised usingstandard procedures.

[0227] Thus, by exchanging the normal, e.g. iodine/pyridine/H₂O,oxidation used for synthesis of phosphordiester oligomers with anoxidation using Beaucage's reagent (commercially available), thephosphorthioate LNA oligomer was efficiently synthesised (stepwisecoupling yields>=98%). The 2′-amino-LNA and 2′methylamino-LNAoligonucleotides (Table 9) were efficiently synthesised (step-wisecoupling yields≧98%) using amidites 74A and 74F. The 2′-thio-LNAoligonucleotides (Table 8) were efficiently synthesised using amidite76F using the standard phosphoramidite procedures as described above forLNA oligonucleotides. After synthesis of the desired sequence, work upwas done using standard conditions (cleavage from solid support andremoval of protection groups using 30% ammonia for 55° C. for 5 h).Purification of LNA oligonucleotides was done using disposable reversedphase purification cartridges and/or reversed phase HPLC and/orprecipitation from ethanol or butanol. Capillary gel electrophoresis,reversed phase HPLC and MALDI-MS was used to verify the purity of thesynthesised oligonucleotide analogues, and to verify that the desirednumber of bicyclic nucleoside analogues of the invention wereincorporated as contemplated.

[0228] An additional aspect of the present invention is to furnishprocedures for oligonucleotide analogues containing LNA linked bynon-natural internucleoside linkages. For example, synthesis of thecorresponding phosphorothioate or phosphoramidate analogues is possibleusing strategies well-established in the field of oligonucleotidechemistry (Protocols for Oligonucleotides and Analogs, vol 20, (SudhirAgrawal, ed.), Humana Press, 1993, Totowa, N.J.; S. L. Beaucage and R.P. Iyer, Tetrahedron, 1993, 49, 6123; S. L. Beaucage and R. P. Iyer,Tetrahedron, 1992, 48, 2223; E. Uhlmann and A. Peyman, Chem. Rev., 90,543).

[0229] Thus, generally the present invention also provides the use of anLNA as defined herein for the preparation of an LNA modifiedoligonucleotides. Is should be understood that LNA modifiedoligonucleotide may comprise normal nucleosides (i.e. naturallyoccurring nucleosides such as ribonucleosides and/ordioxyribonucleosides), as well as modified nucleosides different fromthose defined with the general formula II. In a particularly interestingembodiment, incorporation of LNA modulates the ability of theoligonucleotide to act as a substrate for nucleic acid active enzymes.

[0230] Furthermore, solid support materials having immobilised theretoan optionally nucleobase protected and optionally 5′-OH protected LNAare especially interesting as material for the synthesis of LNA modifiedoligonucleotides where an LNA monomer is included in at the 3′ end. Inthis instance, the solid support material is preferable CPG, e.g. areadily (commercially) available CPG material onto which a3′-functionalised, optionally nucleobase protected and optionally 5′-OHprotected LNA is linked using the conditions stated by the supplier forthat particular material. BioGenex Universial CPG Support (BioGenex,U.S.A.) can e.g. be used. The 5′-OH protecting group may, e.g., be a DMTgroup. 3′-functional group should be selected with due regard to theconditions applicable for the CPG material in question.

[0231] Applications

[0232] The present invention discloses the surprising finding thatvarious novel derivatives of bicyclic nucleoside monomers (LNAs), whenincorporated into oligonucleotides, dramatically increase the affinityof these modified oligonucleotides for both complementary ssDNA andssRNA compared to the unmodified oligonucleotides. It further disclosesthe surprising finding that both fully and partly LNA modifiedoligonucleotides display greatly enhanced hybridisation properties fortheir complementary nucleic acid sequences. Depending on theapplication, the use of these LNAs thus offers the intriguingpossibility to either greatly increase the affinity of a standardoligonucleotide without compromising specificity (constant size ofoligonucleotide) or significantly increase the specificity withoutcompromising affinity (reduction in the size of the oligonucleotide).The present invention also discloses the unexpected finding that LNAmodified oligonucleotides, in addition to greatly enhanced hybridisationproperties, display many of the useful physicochemical properties ofnormal DNA and RNA oligonucleotides. Examples given herein includeexcellent solubility, a response of LNA modified oligonucleotides tosalts like sodium chloride and tetramethylammonium chloride which mimicthat of the unmodified oligonucleotides, the ability of LNA modifiedoligonucleotides to act as primers for a variety of polymerases, theability of LNA modified nucleotides to act as primers in a targetamplification reaction using a thermostable DNA polymerase, the abilityof LNA modified oligonucleotides to act as a substrate for T4polynucleotide kinase, the ability of biotinylated LNAs to sequencespecifically capture PCR amplicons onto a streptavidine coated solidsurface, the ability of immobilised LNA modified oligonucleotides tosequence specifically capture amplicons and very importantly the abilityof LNA modified oligonucleotides to sequence specifically targetdouble-stranded DNA by strand invasion. Hence, it is apparent to one ofordinary skills in the art that these novel nucleoside analogues areextremely useful tools to improve the performance in general ofoligonucleotide based techniques in therapeutics, diagnostics andmolecular biology.

[0233] An object of the present invention is to provide monomeric LNAsaccording to the invention which can be incorporated intooligonucleotides using procedures and equipment well known to oneskilled in the art of oligonucleotide synthesis.

[0234] Another object of the present invention is to provide fully orpartly LNA modified oligonucleotides (oligomers) that are able tohybridise in a sequence specific manner to complementaryoligonucleotides forming either duplexes or triplexes of substantiallyhigher affinity than the corresponding complexes formed by unmodifiedoligonucleotides.

[0235] Another object of the present invention is to use LNAs to enhancethe specificity of normal oligonucleotides without compromisingaffinity. This can be achieved by reducing the size (and thereforeaffinity) of the normal oligonucleotide to an extent that equals thegain in affinity resulting from the incorporation of LNAs.

[0236] Another object of the present invention is to provide fully orpartly modified oligonucleotides containing both LNAs, normalnucleosides and other nucleoside analogues.

[0237] A further object of the present invention is to exploit the highaffinity of LNAs to create modified oligonucleotides of extreme affinitythat are capable of binding to their target sequences in a dsDNAmolecule by way of “strand displacement”.

[0238] A further object of the invention is to provide different classesof LNAs which, when incorporated into oligonucleotides, differ in theiraffinity towards their complementary nucleosides. In accordance with theinvention this can be achieved by either substituting the normalnucleobases G, A, T, C and U with derivatives having, for example,altered hydrogen bonding possibilities or by using LNAs that differ intheir backbone structure. The availability of such different LNAsfacilitates exquisite tuning of the affinity of modifiedoligonucleotides.

[0239] Another object of the present invention is to provide LNAmodified oligonucleotides which are more resistant to nucleases thantheir unmodified counterparts.

[0240] Another object of the present invention is to provide LNAmodified oligonucleotides which can recruit RNAseH.

[0241] An additional object of the present invention is to provide LNAsthat can act as substrates for DNA and RNA polymerases thereby allowingthe analogues to be either incorporated into a growing nucleic acidchain or to act as chain terminators.

[0242] A further object of the present invention is to provide LNAs thatcan act as therapeutic agents. Many examples of therapeutic nucleosideanalogues are known and similar derivatives of the nucleoside analoguesdisclosed herein can be synthesised using the procedures known from theliterature (E. De Clercq, J. Med. Chem. 1995, 38, 2491; P. Herdewijn andE. De Clercq: Classical Antiviral Agents and Design og New AntiviralAgents. In: A Textbook of Drug Design and Development; Eds. P.Krogsgaard-Larsen, T. Liljefors and U. Madsen; Harwood AcademicPublishers, Amsterdam, 1996, p. 425; I. K. Larsen: Anticancer Agents.In: A Textbook of Drug Design and Development; Eds. P.Krogsgaard-Larsen, T. Liljefors and U. Madsen; Harwood AcademicPublishers, Amsterdam, 1996, p. 460).

[0243] Double-stranded RNA has been demonstrated to posses anti-viralactivity and tumour suppressing activity (Sharp et al., Eur. J. Biochem.230(1): 97-103, 1995, Lengyel-P. et al., Proc. Natl. Acad. Sci. U.S.A.,90(13): 5893-5, 1993, and Laurent-Crawford et al., AIDS Res. Hum.Retroviruses, 8(2): 285-90, 1992). It is likely that double strandedLNAs may mimic the effect of therapeutically active double stranded RNAsand accordingly such double stranded LNAs has a potential as therapeuticdrugs.

[0244] When used herein, the term “natural nucleic acid” refers tonucleic acids in the broadest sense, like for instance nucleic acidspresent in intact cells of any origin or vira or nucleic acids releasedfrom such sources by chemical or physical means or nucleic acids derivedfrom such primary sources by way of amplification. The natural nucleicacid may be single, double or partly double stranded, and may be arelatively pure species or a mixture of different nucleic acids. It mayalso be a component of a crude biological sample containing othernucleic acids and other cellular components. On the other hand, the term“synthetic nucleic acids” refers to any nucleic acid produced bychemical synthesis.

[0245] The present invention also provides the use of LNA modifiedoligonucleotides in nucleic acid based therapeutic, diagnostics andmolecular biology. The LNA modified oligonucleotides can be used in thedetection, identification, capture, characterisation, quantification andfragmentation of natural or synthetic nucleic acids, and as blockingagents for translation and transcription in vivo and in vitro. In manycases it will be of interest to attach various molecules to LNA modifiedoligonucleotides. Such molecules may be attachedto either end of theoligonucleotide or they may be attached at one or more internalpositions. Alternatively, they may be attached to the oligonucleotidevia spacers attached to the 5′ or 3′ end. Representative groups of suchmolecules are DNA intercalators, photochemically active groups,thermochemically active groups, chelating groups, reporter groups, andligands. Generally all methods for labelling unmodified DNA and RNAoligonucleotides with these molecules can also be used to label LNAmodified oligonucleotides. Likewise, all methods used for detectinglabelled oligonucleotides generally apply to the corresponding labelled,LNA modified oligonucleotides.

[0246] Therapy

[0247] The term “strand displacement” relates to a process whereby anoligonucleotide binds to its complementary target sequence in a doublestranded DNA or RNA so as to displace the other strand from said targetstrand.

[0248] In an aspect of the present invention, LNA modifiedoligonucleotides capable of performing “strand displacement” areexploited in the development of novel pharmaceutical drugs based on the“antigene” approach. In contrast to oligonucleotides capable of makingtriple helices, such “strand displacement” oligonucleotides allow anysequence in a dsDNA to be targeted and at physiological ionic strengthand pH.

[0249] The “strand displacing” oligonucleotides can also be usedadvantageously in the antisense approach in cases where the RNA targetsequence is inaccessible due to intramolecular hydrogen bonds. Suchintramolecular structures may occur in mRNAs and can cause significantproblems when attempting to “shut down” the translation of the mRNA bythe antisense approach.

[0250] Other classes of cellular RNAs, like for instance tRNAs, rRNAssnRNAs and scRNAs, contain intramolecular structures that are importantfor their function. These classes of highly structured RNAs do notencode proteins but rather (in the form of RNA/protein particles)participate in a range of cellular functions such as mRNA splicing,polyadenylation, translation, editing, maintainance of chromosome endintegrity, etc. Due to their high degree of structure, that impairs oreven prevent normal oligonucleotides from hybridising efficiently, theseclasses of RNAs has so far not attracted interest as antisense targets.

[0251] The use of high affinity LNA monomers should facilitate theconstruction of antisense probes of sufficient thermostability tohybridise effectively to such target RNAs. Therefore, in a preferredembodiment, LNA is used to confer sufficient affinity to theoligonucleotide to allow it to hybridise to these RNA classes therebymodulating the qualitative and/or quantitative function of the particlesin which the RNAs are found.

[0252] In some cases it may be advantageous to down-regulate theexpression of a gene whereas in other cases it may be advantageous toactivate it. As shown by Mollegaard et al. (Mollegaard, N. E.; Buchardt,O.; Egholm, M.; Nielsen, P. E. Proc. Natl. Acad. Sci. U.S.A. 1994, 91,3892), oligomers capable of “strand displacement” an function as RNAtranscriptional activators. In an aspect of the present invention, theLNAs capable of “strand displacement” are used to activate genes oftherapeutic interest.

[0253] In chemotherapy of numerous viral infections and cancers,nucleosides and nucleoside analogues have proven effective. LNAnucleosides are potentially useful as such nucleoside based drugs.

[0254] Various types of double-stranded RNAs inhibit the growth ofseveral types of cancers. Duplexes involving one or more LNAoligonucleotide(s) are potentially useful as such double-stranded drugs.

[0255] The invention also concerns a pharmaceutical compositioncomprising a pharmaceutically active LNA modified oligonucleotide or apharmaceutically active LNA monomer as defined above in combination witha pharmaceutically acceptable carrier.

[0256] Such compositions may be in a form adapted to oral, parenteral(intravenous, intraperitoneal), intramuscular, rectal, intranasal,dermal, vaginal, buccal, ocularly, or pulmonary administration,preferably in a form adapted to oral administration, and suchcompositions may be prepared in a manner well-known to the personskilled in the art, e.g. as generally described in “Remington'sPharmaceutical Sciences”, 17. Ed. Alfonso R. Gennaro (Ed.), MarkPublishing Company, Easton, Pa., U.S.A., 1985 and more recent editionsand in the monographs in the “Drugs and the Pharmaceutical Sciences”series, Marcel Dekker.

[0257] Diagnostics

[0258] Several diagnostic and molecular biology procedures have beendeveloped that utilise panels of different oligonucleotides tosimultaneously analyse a target nucleic acid for the presence of aplethora of possible mutations. Typically, the oligonucleotide panelsare immobilised in a predetermined pattern on a solid support such thatthe presence of a particular mutation in the target nucleic acid can berevealed by the position on the solid support where it hybridises. Oneimportant prerequisite for the successful use of panels of differentoligonucleotides in the analysis of nucleic acids is that they are allspecific for their particular target sequence under the single appliedhybridisation condition. Since the affinity and specificity of standardoligonucleotides for their complementary target sequences depend heavilyon their sequence and size this criteria has been difficult to fulfil sofar.

[0259] In a preferred embodiment, therefore, LNAs are used as a means toincrease affinity and/or specificity of the probes and as a means toequalise the affinity of different oligonucleotides for theircomplementary sequences. As disclosed herein such affinity modulationcan be accomplished by, e.g., replacing selected nucleosides in theoligonucleotide with an LNA carrying a similar nucleobase. As furthershown herein, different classes of LNAs exhibit different affinities fortheir complementary nucleosides. For instance, the 2-3 bridged LNA withthe T-nucleobase exhibits less affinity for the A-nucleoside than thecorresponding 2-4 bridged LNA. Hence, the use of different classes ofLNAs offers an additional level for fine-tuning the affinity of aoligonucleotide.

[0260] In another preferred embodiment the high affinity and specificityof LNA modified oligonucleotides is exploited in the sequence specificcapture and purification of natural or synthetic nucleic acids. In oneaspect, the natural or synthetic nucleic acids are contacted with theLNA modified oligonucleotide immobilised on a solid surface. In thiscase hybridisation and capture occurs simultaneously. The capturednucleic acids may be, for instance, detected, characterised, quantifiedor amplified directly on the surface by a variety of methods well knownin the art or it may be released from the surface, before suchcharacterisation or amplification occurs, by subjecting the immobilised,modified oligonucleotide and captured-nucleic acid to dehybridisingconditions, such as for example heat or by using buffers of low ionicstrength.

[0261] The solid support may be chosen from a wide range of polymermaterials such as for instance CPG (controlled pore glass),polypropylene, polystyrene, polycarbonate or polyethylene and it maytake a variety of forms such as for instance a tube, a micro-titerplate, a stick, a bead, a filter, etc. The LNA modified oligonucleotidemay be immobilised to the solid support via its 5′ or 3′ end (or via theterminus of linkers attached to the 5′ or 3′ end) by a variety ofchemical or photochemical methods usually employed in the immobilisationof oligonucleotides or by non-covalent coupling such as for instance viabinding of a biotinylated LNA modified oligonucleotide to immobilisedstreptavidin. One preferred method for immobilising LNA modifiedoligonucleotides on different solid supports is photochemical using aphotochemically active anthraquinone covalently attached to the 5′ or 3′end of the modified oligonucleotide (optionally via linkers) asdescribed in (WO 96/31557). Thus, the present invention also provide asurface carrying an LNA modified oligonucleotide.

[0262] In another aspect the LNA modified oligonucleotide carries aligand covalently attached to either the 5′ or 3′ end. In this case theLNA modified oligonucleotide is contacted with the natural or syntheticnucleic acids in solution whereafter the hybrids formed are capturedonto a solid support carrying molecules that can specifically bind theligand.

[0263] In still another aspect, LNA modified oligonucleotides capable ofperforming “strand displacement” are used in the capture of natural andsynthetic nucleic acids without prior denaturation. Such modifiedoligonucleotides are particularly useful in cases where the targetsequence is difficult or impossible to access by normal oligonucleotidesdue to the rapid formation of stable intramolecular structures. Examplesof nucleic acids containing such structures are rRNA, tRNA, snRNA andscRNA.

[0264] In another preferred embodiment, LNA modified oligonucleotidesdesigned with the purpose of high specificity are used as primers in thesequencing of nucleic acids and as primers in any of the several wellknown amplification reactions, such as the PCR reaction. As shownherein, the design of the LNA modified oligonucleotides determineswhether it will sustain a exponential or linear target amplification.The products of the amplification reaction can be analysed by a varietyof methods applicable to the analysis of amplification productsgenerated with normal DNA primers. In the particular case where the LNAmodified oligonucleotide primers are designed to sustain a linearamplification the resulting amplicons will carry single stranded endsthat can be targeted by complementary probes without denaturation. Suchends could for instance be used to capture amplicons by othercomplementary LNA modified oligonucleotides attached to a solid surface.

[0265] In another aspect, LNA modified oligos capable of “stranddisplacement” are used as primers in either linear or exponentialamplification reactions. The use of such oligos is expected to enhanceoverall amplicon yields by effectively competing with ampliconre-hybridisation in the later stages of the amplification reaction.Demers, et al. (Nucl. Acid Res. 1995, Vol 23, 3050-3055) discloses theuse of high-affinity, non-extendible oligos as a means of increasing theoverall yield of a PCR reaction. It is believed that the oligomerselicit these effect by interfering with amplicon re-hybridisation in thelater stages of the PCR reaction. It is expected that LNA modifiedoligos blocked at their 3′ end will provide the same advantage. Blockingof the 3′ end can be achieved in numerous ways like for instance byexchanging the 3′ hydroxyl group with hydrogen or phosphate. Such 3′blocked LNA modified oligos can also be used to selectively amplifyclosely related nucleic acid sequences in a way similar to thatdescribed by Yu et al. (Biotechniques, 1997, 23, 714-716).

[0266] In recent years, novel classes of probes that can be used in forexample real-time detection of amplicons generated by targetamplification reactions have been invented. One such class of probeshave been termed “Molecular Beacons”. These probes are synthesised aspartly self-complementary oligonucleotides containing a fluorophor atone end and a quencher molecule at the other end. When free in solutionthe probe folds up into a hairpin structure (guided by theself-complimentary regions) which positions the quencher in sufficientcloseness to the fluorophor to quench its fluorescent signal. Uponhybridisation to its target nucleic acid, the hairpin opens therebyseparating the fluorophor and quencher and giving off a fluorescentsignal.

[0267] Another class of probes have been termed “Taqman probes”. Theseprobes also contain a fluorophor and a quencher molecule. Contrary tothe Molecular Beacons, however, the quenchers ability to quench thefluorescent signal from the fluorophor is maintained after hybridisationof the probe to its target sequence. Instead, the fluorescent signal isgenerated after hybridisation by physical detachment of either thequencher or fluorophor from the probe by the action of the 5′exonuxleaseactivity of a polymerase which has initiated synthesis from a primerlocated 5′ to the binding site of the Taqman probe.

[0268] High affinity for the target site is an important feature in bothtypes of probes and consequently such probes tends to be fairly large(typically 30 to 40 mers). As a result, significant problems areencountered in the production of high quality probes. In a preferredembodiment, therefore, LNA is used to improve production and subsequentperformance of Taqman probes and Molecular Beacons by reducing theirsize whilst retaining the required affinity.

[0269] In a further aspect, LNAs are used to construct new affinitypairs (either fully or partially modified oligonucleotides). Theaffinity constants can easily be adjusted over a wide range and a vastnumber of affinity pairs can be designed and synthesised. One part ofthe affinity pair can be attached to the molecule of interest (e.g.proteins, amplicons, enzymes, polysaccharides, antibodies, haptens,peptides, PNA, etc.) by standard methods, while the other part of theaffinity pair can be attached to e.g. a solid support such as beads,membranes, micro-titer plates, sticks, tubes, etc. The solid support maybe chosen from a wide range of polymer materials such as for instancepolypropylene, polystyrene, polycarbonate or polyethylene. The affinitypairs may be used in selective isolation, purification, capture anddetection of a diversity of the target molecules mentioned above.

[0270] The principle of capturing an LNA-tagged molecule by ways ofinteraction with another complementary LNA oligonucleotide (either fullyor partially modified) can be used to create an infinite number of novelaffinity pairs.

[0271] In another preferred embodiment the high affinity and specificityof LNA modified oligonucleotides are exploited in the construction ofprobes useful in in-situ hybridisation. For instance, LNA could be usedto reduce the size of traditional DNA probes whilst maintaining therequired affinity thereby increasing the kinetics of the probe and itsability to penetrate the sample specimen. The ability of LNA modifiedoligonucleotides to “strand invade” double stranded nucleic acidstructures are also of considerable advantage in in-situ hybridisation,because it facilitates hybridisation without prior denaturation of thetarget DNA/RNA.

[0272] In another preferred embodiment, LNA modified oligonucleotides tobe used in antisense therapeutics are designed with the dual purpose ofhigh affinity and ability to recruit RNAseH. This can be achieved by,for instance, having LNA segments flanking an unmodified central DNAsegment.

[0273] The present invention also provides a kit for the isolation,purification, amplification, detection, identification, quantification,or capture of natural or synthetic nucleic acids, where the kitcomprises a reaction body and one or more LNA modified oligonucleotides(oligomer) as defined herein. The LNA modified oligonucleotides arepreferably immobilised onto said reactions body.

[0274] The present invention also provides a kit for the isolation,purification, amplification, detection, identification, quantification,or capture of natural or synthetic nucleic acids, where the kitcomprises a reaction body and one or more LNAs as defined herein. TheLNAs are preferably immobilised onto said reactions body (e.g. by usingthe immobilising techniques described above).

[0275] For the kits according to the invention, the reaction body ispreferably a solid support material, e.g. selected from borosilicateglass, soda-lime glass, polystyrene, polycarbonate, polypropylene,polyethylene, polyethyleneglycol terephthalate, polyvinylacetate,polyvinylpyrrolidinone, polymethylmethacrylate and polyvinylchloride,preferably polystyrene and polycarbonate. The reaction body may be inthe form of a specimen tube, a vial, a slide,

[0276] a sheet, a film, a bead, a pellet, a disc, a plate, a ring, arod, a net, a filter, a tray, a microtitre plate, a stick, or amulti-bladed stick.

[0277] The kits are typically accompanied by a written instruction sheetstating the optimal conditions for use of the kit.

[0278] The above-mentioned diagnostic and therapeutic aspects of thepresent invention have been illustrated with the following examples.

Example 106

[0279]1-(3,5-Di-O-benzyl-4-C-(p-toulenesulfonyloxymethyl)-β-D-ribofuranosyl)thymine(80). Nucleoside 79 (13.1 g, 19.7 mmol) was dissolved in a solution ofammonia in methanol (200 cm³, prepared by diluting saturated methanolicammonia with an equal volume of methanol) and stirred at roomtemperature for 4 h. The reaction mixture was subsequently evaporated,and the residue was dissolved in dichloromethane (400 cm³). The organicphase was washed with brine (3×150 cm³), dried (Na₂SO₄), filtered andevaporated under reduced pressure. The residue was purified by silicagel column chromatography using dicloromethane:methanol (99.5:0.5, v/v)as eluent to give nucleoside 80 as a white solid material (10.7 g, 87%).δ_(H) (CDCl₃) 9.66 (s, 1H, NH), 7.71-7.21 (15H, m, 6-H, aromatic), 5.72(1H, d, J 5.1, 1′-H), 4.75, 4.55 (2H, each d, J 11.5, Bn), 4.51 (2H, s,Bn), 4.37 (1H, t, J 5.4, 2′-H), 4.30-4.12 (3H, m, 3′-H, Bn), 3.76 (1H,d, J 10.2, 1″-H_(a)), 3.59 (1H, d, J 10.2, 1″-H_(b)), 2.39 (3H, s,CH₃C₆H₅), 1.48 (3H, s, CH₃). δ_(C) (CDCl₃) 163.8 (C-4), 150.9 (C-2),145.0, 137.0, 136.9, 135.9, 132.3, 129.8, 128.7, 128.6, 128.2, 128.1,128.0, 127.6 (aromatic), 111.0 (C-5), 89.6, 85.3, 78.4, 74.5, 73.8,71.1, 69.7, (Bn, C-1′, C-3′, C-2′, C-4′, C-1″), 21.6 (CH₃), 12.0 (CH₃).FAB-MS m/z 623 [M+H]⁺ (Found C, 61.5; H, 5.2; N, 4.4; S, 5.2,C₃₂H₃₄O₉N₂S requires C, 61.7; H, 5.4; N, 4.5; S, 5.1).

Example 107

[0280](1S,3R,4R,7S)-7-Benzyloxy-1-benzoyloxymethyl-3-(thymin-1-yl)-2,5-dioxabicyclo-[2.2.1]heptane(36). To a stirred solution of nucleoside 80 (10.65 g, 17.1 mmol) inanhydrous DMF (150 cm³) was added a 60% suspension of sodium hydride inmineral oil (0.9 g, 22.2 mmol) in small portions at 0° C. The mixturewas stirred at 0° C. for 15 h whereupon additional 60% sodium hydride(0.205 g, 5.12 mmol) was added, and the reaction mixture was stirred foradditional 22 h at 0° C. Methanol (20 cm³) was added and the reactionmixture was subsequently concentrated under reduced pressure to halfvolume. Ice-cold H₂O (300 cm³) was added and extraction was performedwith dichloromethane (5×150 cm³). The combined organic phase was washedwith saturated aqueous solutions of sodium hydrogen carbonate (3×40 cm³)and brine (3×40 cm³), dried (Na₂SO₄), filtered and evaporated underreduced pressure. The residue was purified by silica gel columnchromatography using dichloromethane:methanol (99.5:0.5, v/v) as eluentto give nucleoside 36 as a white solid material (7.1 g, 92%). Spectraldata were in accordance with data given earlier for 36 (Found C, 66.2;H, 5.8; N, 6.1; C₂₅H₂₆N₂O₆ requires C, 66.6; H, 5.8; N, 6.2).

Example 108

[0281]3,5-Di-O-benzyl-1,2-O-isopropylidene-4-C-methanesulfonyloxymethyl-α-D-ribofuranose(200). To a stirred solution of furanose 31 (2.16 g, 5.39 mmol) inanhydrous pyridine (3 mL) at 0° C. was added dropwise methanesulfonylchloride (0.61 mL, 16.0 mmol). The reaction mixture was stirred for 20min at room temperature, quenched with ice-cold water (300 mL) andextracted with dichloromethane (2×300 mL). The combined extracts werewashed with saturated aqueous sodium hydrogen carbonate (300 mL) andthen dried (MgSO₄). The solvent was removed by distillation underreduced pressure and the residue was purified-by chromatography oversilica gel with dichloromethane as eluent to give the product 200 as aclear oil (2.55 g, 99%); ¹H NMR (CDCl₃): δ 7.37-7.24 (10H, m, Bn), 5.78(1H, d, J 3.8 Hz, H-1), 4.85 (1H, d, J 11.7 Hz, Bn), 4.73 (1H, d, J 11.9Hz, Bn), 4.64 (1H, dd, J 4.0, 5.3 Hz, H-2), 4.54 (1H, d, J 11.9 Hz,H-5′), 4.52 (1H, d, J 11.9 Hz, Bn), 4.46 (1H, d, J 11.9 Hz, H-5′), 4.41(1H, d, J 11.8 Hz, Bn), 3.60 (1H, d, J 10.4 Hz, H-5), 3.50 (1H, d, J10.5 Hz, H-5), 3.06 (3H, s, SO₂CH₃), 1.68 (3H, s, CH₃), 1.34 (3H, s,CH₃); ¹³C NMR (CDCl₃): δ 137.79, 137.31, 128.54, 128.48, 128.16, 128.01,127.87, 127.79 (Bn), 113.66 (C(CH₃)₂), 104.46 (C-1), 84.88 (C-4), 78.48,78.41 (C-2, C-3), 73.65, 72.63, 70.78, 70.16 (Bn, C-5, C-5′), 37.84(SO₂CH₃), 26.20 (CH₃), 25.69 (CH₃); MS FAB: 501 (M+Na, 100%). Found: C,60.37; H, 6.29; S, 6.53; C₂₄H₃₀O₈S requires C, 60.24; H, 6.32; S, 6.70%.

Example 109

[0282] Methyl3,5-di-O-benzyl-4-C-methanesulfonyloxymethyl-α-D-ribofuranoside (201). Asolution of furanose 200 (1.133 g, 2.37 mmol) in methanolic hydrochloricacid (20% w/w, 31.7 mL) and water (4.4 mL) was stirred at roomtemperature for 2 h. After neutralisation with sodium hydrogen carbonate(s), the solution was extracted with dichloromethane (2×150 mL). Thecombined extracts were washed with water (150 mL) and then dried(MgSO₄). The solvent was removed by distillation under reduced pressureand the residue purified by chromatography over silica gel withdichloro-methane:methanol (99:1) as eluent to give the product 201(β:α˜2:1) as a clear oil (1.018 g, 95%); ¹H NMR (CDCl₃): δ 7.39-7.22 (m,Bn), 4.86 (br s, Bn), 4.69-3.99 (m, Bn, H-5′, H-1, H-2, H-3), 3.68 (d, J8.9 Hz, H-5 β), 3.51 (d, J 9.8 Hz, H-5 α), 3.46 (s, OCH₃ α), 3.34 (d, J9.1 Hz, H-5 β), 3.32 (d, J 9.7 Hz, H-5 α), 3.28 (s, OCH₃ β), 2.97 (3H,s, SO₂CH₃ β), 2.93 (3H, s, SO₂CH₃ α); ¹³C NMR (CDCl₃): δ 137.74, 136.98,128.70, 128.64, 128.58, 128.56, 128.37, 128.21, 128.15, 128.09, 127.98,127.86, 127.83 (Bn), 107.54 (C-1 β), 103.39 (C-1 α), 84.65, 83.18,81.90, 78, 87 (C-4, C-3), 75.04, 74.07, 73.73, 73.70, 73.38, 72.56,72.11, 70.85, 70.55, 70.20 (C-2, Bn, C-5, C-5′), 55.90 (OCH₃ α), 54.96(OCH₃ β), 37.18 (SO₂CH₃ β), 37.07 (SO₂CH₃ α); MS FAB: 475 (M+Na, 25%).Found: C, 58.40; H, 6.33; C₂₄H₃₀O₈S requires C, 58.39; H, 6.24%.

Example 110

[0283] (3R)- and(3S)-(1S,4R,7S)-7-Benzyloxy-1-benzyloxymethyl-3-methoxy-2,5-dioxabicyclo[2.2.1]heptane(202 and 203). A solution of 201 (3.32 g, 7.34 mmol) in anhydrous DMF(25 mL) was stirred at 0° C. and a 60% oil dispersion of sodium hydride(700 mg, 16.9 mmol) was added. The mixture was stirred at roomtemperature for 90 min, quenched with water (300 mL) and extracted withdiethyl ether (2×300 mL). The combined extract was washed with water(200 mL) and dried (MgSO₄). The solvent was removed under reducedpressure and the residue was purified by chromatography over silica gelwith dichloromethane as eluent to give the two products 202 and 203 asclear oils (1.571 g, 60% and 0.777 g, 30% respectively).(1S,3R,4R,7S)-7-Benzyloxy-1-benzyloxymethyl-3-methoxy-2,5-dioxabicyclo[2.2.1]heptane(202). ¹H NMR (CDCl₃): δ 7.36-7.26 (10H, m, Bn), 4.81 (1H, s, H-1), 4.65(1H, d, J 11.9 Hz, Bn), 4.61 (2H, s, Bn), 4.56 (1H, d, J 11.9 Hz, Bn),4.11 (1H, s, H-2), 4.09 (1H, s, H-3), 4.01 (1H, d, J 7.5 Hz, H-5′),3.80-3.77 (3H, m, H-5′, H-5), 3.39 (3H, s, OCH₃); ¹³C NMR (CDCl₃): δ138.05, 137.36, 128.47, 128.44, 127.88, 127.73, 127.63 (Bn), 104.97(C-1), 85.13 (C-4), 79.16 (C-3), 77.18 (C-2), 73.64 (Bn), 72.26, 72.10(Bn, C-5′), 66.50 (C-5), 55.34 (OCH₃); MS FAB: 379 (M+Na, 28%). Found:C, 70.55; H, 6.97; C₂₁H₂₄O₅ requires C, 70.77; H, 6.79%.(1S,3R,4R,7S)-7-Benzyloxy-1-benzyloxymethyl-3-methoxy-2,5-dioxabicyclo-[2.2.1]heptane(203). ¹H NMR (CDCl₃): δ 7.36-7.26 (10H, m, Bn), 5.00 (1H, s, H-1),4.67-4.54 (4H, m, Bn), 4.18 (1H, s, H-2), 3.99 (1H, s, H-3), 3.99-3.90(2H, m, H-5′), 3.75-3.68 (2H, m, H-5), 3.49 (3H, s, OCH₃); ¹³C NMR(CDCl₃): δ 137.83, 137.53, 128.51, 128.48, 127.96, 127.82, 127.71,127.62 (Bn), 104.05 (C-1), 88.44 (C-4), 79.54 (C-3), 77.16 (C-2), 73.68(Bn), 72.61 (C-5′), 72.24 (Bn), 65.73 (C-5), 56.20 (OCH₃); MS FAB: 379(M+Na, 100%).

Example 111

[0284](1R,2S,3S)-2-Benzyloxy-3-benzyloxymethyl-1-(methoxy(thymin-1-yl)methyl)-3-trimethylsilyloxytetrahydrofuran(204). A solution of 202 (216 mg, 0.606 mmol) and thymine (153 mg, 1.22mmol) in anhydrous acetonitrile (9.3 mL) was added BSA(N,O-bis(trimethylsilyl)acetamide, 0.90 mL, 3.6 mmol) and stirred underreflux for 15 min. The solution was cooled to 0° C. and trimethylsilyltriflate (0.153 mL, 0.777 mmol) was added dropwise. After stirring atroom temperature for 18 h and at 60° C. for 24 h, the reaction wasquenched with a saturated aqueous solution of sodium hydrogen carbonate(20 mL), and extraction was performed using dichloromethane (2×50 mL).The combined extract was washed with a saturated aqueous solution ofsodium hydrogen carbonate (50 mL) and dried (MgSO₄). The solvent wasremoved under reduced pressure and the residue was purified bychromatography over silica gel with dichloromethane:methanol (98:2) aseluent to give the product 204 (mixture of diastereomers˜1.7:1) as asolid (196 mg, 67%). ¹H NMR (CDCl₃): δ 7.36-7.14 (m, Bn, H-6), 5.77 (1H,d, J 7.9 Hz, H-1′), 5.57 (1H, d, J 5.8 Hz, H-1′), 4.68-4.43 (m, Bn,H-2′), 4.12-3.68 (m, H-5′, H-5′, H-3′), 3.32 (s, OCH₃), 3.24 (s, OCH₃),1.93 (d, J 0.9 Hz, CH₃), 1.86 (d, J 1.1 Hz, CH₃), 0.14 (s, Si(CH₃)₃),0.12 (s, Si(CH₃)₃); ¹³C NMR (CDCl₃): δ 163.68, 163.55 (C-4), 151.58,151.07 (C-2), 137.84, 137.74, 137.32 (Bn), 135.93, 135.10 (C-6), 128.57,128.42, 128.41, 128.10, 127.95, 127.85, 127.77, 127.74 (Bn), 111.38,111.01 (C-5), 86.89, 85.61, 85.40, 84.72, 83.40, 83.31, 82.10 (C-1′,C-2′, C-3′, C-4′), 75.20, 73.98, 73.62, 73.59, 72.55, 72.13, 71.04,70.74 (Bn, C-5′, C-5″), 56.82, 56.54 (OCH₃), 12.47, 12.3-8 (CH₃), 1.72,1.69 (Si(CH₃)₃); MS FAB: 555 (M+H, 65%), 577 (M+Na, 70%). Found: C,62.76; H, 6.88; N, 4.94; C₂₉H₃₈N₂O₇Si requires C, 62.79; H, 6.90; N,5.05%.

Example 112

[0285](1R,2S,3S)-2-Benzyloxy-3-benzyloxymethyl-1-(methoxy(6-N-benzoyladenin-9-yl)methyl)-3-trimethylsilyloxytetrahydrofuran(205). A solution of 202 (240 mg, 0.673 mmol) and 6-N-benzoyladenine(301 mg, 1.26 mmol) in anhydrous acetonitrile (8.2 mL) was added BSA(0.67 mL, 2.7 mmol) and stirred at room temperature for 1 h. Thesolution was cooled to 0° C. and trimethylsilyl triflate (0.25 mL, 1.33mmol) was added dropwise. After stirring at 65° C. for 18 h, thereaction was quenched with a saturated aqueous solution of sodiumhydrogen carbonate (50 mL), extracted with dichloromethane (2×50 mL).The combined extract was dried (MgSO₄). The solvent was removed underreduced pressure and the residue was purified by chromatography oversilica gel with dichloromethane:methanol (98:2) as eluent to give theproduct 205 (mixture of diastereomers˜1.8:1) as a solid (185 mg, 41%).¹H NMR (CDCl₃): δ 8.78 (s, H-8), 8.21 (s, H-2), 8.17 (s, H-2), 8.03-8.00(m, Bz), 7.61-7.49 (m, Bz), 7.36-7.23 (m, Bn), 7.07-7.04 (m, Bz), 5.85(1H, d, J 7.9 Hz, H-1′), 5.76 (1H, d, J 6.0 Hz, H-1′), 4.74-4.40 (m, Bn,H-2′), 4.22-3.62 (m, H-5′, H-5′, H-3′), 3.33 (s, OCH₃), 3.24 (s, OCH₃),0.15 (s, Si(CH₃)₃), 0.14 (s, Si(CH₃)₃); ¹³C NMR (CDCl₃): δ 164.68(HNC═O), 153.17, 152.99 (C-6), 149.47 (C-2), 141.82, 141.66 (C-8),137.74, 137.71, 137.65 (Bn), 133.87, 132.87, 132.78 (Bz), 128.97,128.93, 128.45, 128.42, 128.38, 128.14, 127.97, 127.88, 127.82, 127.78(Bn, Bz), 123.66, 122.85 (C-5), 86.41, 86.23, 85.70, 85.24, 84.78,83.73, 83.58, 82.79 (C-1′, C-2′, C-3′, C-4′), 75.32, 74.55, 73.61,72.18, 71.98, 70.85, 70.59 (Bn, C-5′, C-5″), 57.23, 57.04 (OCH₃), 1.78(Si(CH₃)₃); MS FAB: 668 (M+H, 50%), 690 (M+Na, 100%). Found: C, 64.07;H, 6.01; N, 9.94; C₂₉H₃₈N₂O₇Si, 0.5H₂O requires C, 63.88; H, 6.25; N,10.34%.

Example 113

[0286](1R,2R,3R)-2-Benzyloxy-3-benzyloxymethyl-3-hydroxytetrahydrofurfural(206). A solution of 202/203 (252 mg, 0.707 mmol) in 80% acetic acid(3.8 mL) was stirred at 90° C. for 2 h whereupon the solvent was removedby distillation under reduced pressure. The residue was coevaporated intoluene (3×10 mL) to give the product 206 as an oil (242 mg, 100%). ¹HNMR (CDCl₃): δ 9.66 (1H, d, J 0.8 Hz, H-1), 7.36-7.25 (10H, m, Bn), 4.68(1H, d, J 11.9 Hz, Bn), 4.60-4.39 (5H, m, Bn, H-2, H-3), 3.98-3.92 (2H,m, H-5), 3.85 (1H, d, J 9.3 Hz, H-5′), 3.52 (1H, d, J 9.2 Hz, H-5′); ¹³CNMR (CDCl₃): δ 203.64 (C-1), 137.39, 137.19, 128.61, 128.54, 128.29,128.12, 127.87, 127.83 (Bn), 87.17, 87.05 (C-4, C-2), 80.98 (C-3),75.00, 73.70, 71.86 (Bn, C-5′), 67.84 (C-5); MS FAB: 707 (2×M+Na, 100%).

Example 114

[0287](1S,3S,4R,7S)-3-Acetoxy-7-benzyloxy-1-benzyloxymethyl-2,5-dioxabicyclo[2.2.1]-heptane(207). To a stirred solution of 206 (230 mg, 0.672 mmol) in anhydrouspyridine (2.0 mL) was added acetic anhydride (0.18 mL, 1.91 mmol). Thereaction mixture was stirred for 23 h at room temperature, water (0.13mL) was added, and the solvent was removed by distillation under reducedpressure. The residue was coevaporated in toluene (3×10 mL) and purifiedby chromatography over silica gel with dichloromethane:methanol (99:1)as eluent to give the product 207 as an clear oil (56.7 mg, 23%); ¹H NMR(CDCl₃): δ 7.38-7.26 (10H, m, Bn), 6.00 (1H, s, H-1), 4.68 (1H, d, J12.0 Hz, Bn), 4.62 (1H, d, J 12.2 Hz, Bn), 4.60 (1H, d, J 12.4 Hz, Bn),4.56 (1H, d, J 12.2 Hz, Bn), 4.17 (1H, s, H-2), 4.14 (1H, s, H-3), 4.01(1H, d, J 7.7 Hz, H-5′), 3.81-3.78 (3H, m, H-5′, H-5), 20.06 (3H, s,COCH₃); ¹³C NMR (CDCl₃): δ 169.18 (CO), 137.92, 137.48, 128.52, 128.45,128.03, 127.77, 127.73, 127.68 (Bn), 95.95 (C-1), 86.49 (C-4), 78.27,76.58 (C-3, C-2), 73.65 (Bn), 72.26, 71.96 (Bn, C-5′), 65.49 (C-5),20.98 (COCH₃); MS FAB: 407 (M+Na, 55%). Found: C, 68.80; H, 6.11;C₂₂H₂₄O₆ requires C, 68.74; H, 6.29%.

Example 115

[0288](1S,3S,4R,7S)-3-(6-N-Benzoyladenin-9-yl)-7-benzyloxy-1-benzyloxymethyl-2,5-dioxabicyclo[2.2.1]heptane(208). A solution of furanose 207 (167 mg, 0.434 mmol) and6-N-benzoyladenine (194 mg, 0.813 mmol) in anhydrous acetonitrile (5.3mL) was added BSA (0.43 mL, 1.76 mmol) and stirred at room temperaturefor 1 h. The solution was cooled to 0° C. and trimethylsilyl triflate(0.16 mL, 0.86 mmol) was added dropwise. After stirring at 65° C. for 2h, the reaction was quenched with a saturated aqueous solution of sodiumhydrogen carbonate (40 mL) and the mixture was extracted withdichloromethane (2×50 mL). The combined extract was dried (MgSO₄). Thesolvent was removed under reduced pressure and the residue was purifiedby chromatography over silica gel with dichloromethane:methanol (98:2)as eluent to give the product 208 as a solid (111 mg, 45%); ¹H NMR(CDCl₃): δ 8.82 (1H, s, H-8), 8.14 (1H, s, H-2), 7.59-7.26 (15H, m, Bz,Bn), 6.74(1H, s, H-1′), 4.92 (1H, s, H-2′), 4.74-4.39 (4H, m, Bn), 4.42(1H, s, H-3′), 4.19-4.10 (2H, m, H-5″), 3.92 (1H, d, J 11.8 Hz, H-5′),3.88 (1H, d, J 11.5 Hz, H-5′); MS FAB: 564 (M+H, 100%).

Example 116

[0289] Methyl2-O-acetyl-3,5-di-O-benzyl-4-C-methanesulfonyloxymethyl-D-ribofuranoside(209). To a stirred solution of 201 (687 mg, 1.52 mmol) in anhydrouspyridine (4 mL) at 0° C. was added dropwise acetic anhydride (0.43 mL,4.56 mmol). The reaction mixture was stirred for 2 days at roomtemperature, quenched with saturated aqueous sodium hydrogen carbonate(75 mL) and extracted with dichloromethane (150+75 mL). The combinedextract was dried (MgSO₄), the solvent was removed by distillation underreduced pressure and the residue was purified by chromatography oversilica gel with dichloromethane as eluent to give the product 209 as aclear oil (β:α˜3:1, 750 mg, 100%); MS FAB: 463 (M-OCH₃, 100%), 517(M+Na, 28%); Found: C, 58.53; H, 6.16; C₂₄H₃₀O₉S requires C, 58.29; H,6.11%. Methyl2-O-acetyl-3,5-di-O-benzyl-4-C-methanesulfonyloxymethyl-β-D-ribofuranoside(209β). ¹H NMR (CDCl₃): δ 7.36-7.18 (10H, m, Bn), 5.27 (1H, d, J 4.9 Hz,H-2), 4.88 (1H, s, H-1), 4.55-4.44 (6H, m, H-5′, Bn), 4.35 (1H, d, J 5.0Hz, H-3), 3.73 (1H, d, J 9.2 Hz, H-5), 3.38 (1H, d, J 9.3 Hz, H-5), 3.30(3H, s, OCH₃), 2.95 (3H, s, SO₂CH₃), 2.11 (3H, s, OCCH₃); ¹³C NMR(CDCl₃): δ 169.91 (C═O), 137.83, 137.28, 128.49, 128.44, 127.99, 127.87,127.77 (Bn), 105.40 (C-1), 82.65, 81.05, 74.55, 73.62, 73.56, 71.86,70.22 (C-2, C-3, C-4, C-5, C-5′, Bn), 55.03 (OCH₃), 37.14 (SO₂CH₃),20.73 (OCCH₃). Methyl2-O-acetyl-3,5-di-O-benzyl-4-C-methanesulfonyloxymethyl-α-D-ribofuranoside(209a). ¹H NMR (CDCl₃): δ 7.36-7.18 (10H, m, Bn), 5.09 (1H, d, J 4.5 Hz,H-1), 4.95 (1H, dd, J 4.5, 6.8 Hz, H-2), 4.65-4.44 (6H, m, H-5′, Bn),4.27 (1H, d, J 6.6 Hz, H-3), 3.49 (1H, d, J 9.9 Hz, H-5), 3.46 (3H, s,OCH₃), 3.36 (1H, d, J 9.9 Hz, H-5), 2.92 (3H, S, SO₂CH₃), 2.14 (3H, s,OCCH₃); ¹³C NMR (CDCl₃): δ 170.41 (C═O), 137.59, 137.28, 128.56, 128.51,128.49, 128.44, 127.98, 127.88 (Bn), 102.35 (C-1), 84.25, 77.53, 74.66,73.67, 72.12, 70.39, 70.28 (C-2, C-3, C-4, C-5, C-5′, Bn), 56.07 (OCH₃),36.94 (SO₂CH₃), 20.63 (OCCH₃).

Example 117

[0290] Phenyl2-O-acetyl-3,5-di-O-benzyl-4-C-methanesulfonyloxymethyl-1-thio-p-D-ribofuranoside(210). Method a. A stirred solution of 209 (738 mg, 1.49 mmol) inanhydrous dichloromethane (6.4 mL) was added phenylthiotrimethylsilane(2.42 mL, 12.8 mmol) and cooled to 0° C. Trimethylsilyl triflate (0.67mL, 3.67 mmol) was added dropwise and the solution was stirred at roomtemperature for 4 h. The reaction was quenched with a saturated aqueoussolution of sodium hydrogen carbonate (100 mL) and extracted withdichloromethane (2×200 mL). The combined extract was dried (MgSO₄) andthe solvent removed by distillation under reduced pressure. The residuewas purified by chromatography over silica gel with dichloromethane aseluent to give the product 210 as a clear oil (564 mg, 66%) andunreacted starting material (191 mg, 26%); Method b. A stirred solutionof 211 (86 mg, 0.165 mmol) in anhydrous dichloromethane (0.49 mL) wasadded phenylthiotrimethylsilane (0.16 mL, 0.825 mmol) and cooled to 0°C. Trimethylsilyl triflate (0.037 mL, 0.206 mmol) was added and thesolution was stirred at room temperature for 2 h The reaction wasquenched with a saturated aqueous solution of sodium hydrogen carbonate(15 mL) and the resulting mixture was extracted with dichloromethane(2×25 mL). The combined extract was dried (MgSO₄) and the solventremoved by distillation under reduced pressure. The residue was purifiedby chromatography over silica gel with dichloromethane as eluent to givethe product 210 as a clear oil (75 mg, 79%); ¹H NMR (CDCl₃): δ 7.47-7.19(15H, m, Bn, SPh), 5.48 (1H, d, J 3.6 Hz, H-2), 5.34 (1H, dd, J 3.7, 5.2Hz, H-1), 4.54-4.36 (7H, m, H-3, H-5′, Bn), 3.66 (1H, d, J 9.7 Hz, H-5),3.48 (1H, d, J 9.5 Hz, H-5), 2.89 (3H, s, SO₂CH₃), 2.09 (3H, s, OCCH₃);¹³C NMR (CDCl₃): δ 169.93 (C═O), 137.69, 137.08, 132.65, 132.45, 129.15,128.53, 128.52, 128.18, 128.14, 128.08, 127.91, 127.85 (Bn, SPh), 87.99,84.35, 80.34, 75.33, 74.20, 73.67, 70.83, 69.34 (C-1, C-2, C-3, C-4,C-5, C-5′, Bn), 37.27 (SO₂CH₃), 20.68 (OCCH₃); MS FAB: 463 (M-SPh,100%), 595 (M+Na, 24%); Found: C, 61.17; H, 5.55; C₂₉H₃₂O₈S₂ requires C,60.82; H, 5.63%.

Example 118

[0291]1,2-Di-O-acetyl-3,5-di-O-benzyl-4-C-methanesulphonyloxymethyl-D-ribofuranose(211). A solution of 201 (150 mg; 0.313 mmol) in 80% aqueous acetic acid(1.5 mL) was stirred at 90° C. for 3 h. The solvent was removed bydistillation under reduced pressure and the residue was coevaporated inethanol (3×5 mL), toluene (3×5 mL) and pyridine (2×5 mL). The residuewas redissolved in anhydrous pyridine (0.62 mL) and added aceticanhydride (0.47 mL) and the solution was stirred at room temperature for16 h. The reaction was quenched with water (50 mL) and the resultingmixture extracted with dichloromethane (2×50 mL). The combined extractwas washed with an aqueous saturated solution of sodium hydrogencarbonate (50 mL) and dried (MgSO₄). The solvent was evaporated and theresidue purified on column chromatography over silica gel withdichloromethane as eluent to give the product 211 as an oil (99 mg,60%); ¹H NMR (CDCl₃): δ 7.39-7.21 (m, Bn), 6.38 (d, J 4.6 Hz, H-1 β),6.15 (s, H-1 α), 5.35 (d, J 4.9 Hz, H-2 α), 5.17 (dd, J 6.3, 4.9 Hz, H-2β), 4.69-4.23 (m, H-3, Bn), 3.64 (d, J 9.7 Hz, H-5 α), 3.52 (d, J 10.1Hz, H-2 β), 3.45 (d, J 9.7 Hz, H-5 α), 3.39 (d, J 9.9 Hz, H-2 β), 2.99(S, SO₂CH₃ α), 2.96 (S, SO₂CH₃ β), 2.14, 2.13, 2.06, 1.90 (4×s, COCH₃);¹³C NMR (CDCl₃): δ 169.68, 169.00 (C═O), 137.68, 137.05, 128.60, 128.55,128.50, 128.21, 128.12, 128.04, 127.94, 127.82, 127.79 (Bn), 99.35 (C-1α), 94.24 (C-1 β), 86.36 (C-4 β), 84.28 (C-4 α), 79.15, 77.47, 74.58,74.06, 73.73, 73.56, 71.67, 70.57, 70.19, 69.84 (Bn, C-2, C-3, C-5,C-5′), 37.61 (SO₂CH₃ β), 37.48 (SO₂CH₃α), 21.07, 20.74, 20.63, 20.39(COCH₃); MS FAB: 545 (M+Na, 13%). Found: C, 57.70; H, 5.56; C₂₅H₃₀O₁₀Srequires C, 57.46; H, 5.79%.

Example 119

[0292] (3R)- and(3S)-(1S,4R,7S)-7-Benzyloxy-1-benzyloxymethyl-3-phenylthio-2,5-dioxabicyclo[2.2.1]heptane(212). A solution of 210 (553 mg, 0.966 mmol) in methanol saturated withammonia (35 mL) was stirred at room temperature for 2 h whereupon thesolvent removed by distillation under reduced pressure. The residue wasredissolved in anhydrous DMF (3.5 mL) and the solution stirred at 0° C.A 60% suspension of sodium hydride (118 mg, 2.88 mmol) was added and themixture stirred at room temperature for 12 h. The reaction was quenchedwith a saturated aqueous solution of sodium hydrogen carbonate (100 mL)and the resulting mixture was extracted with dichloromethane (2×100 mL).The combined extract was dried (MgSO₄) and the solvent was removed bydistillation under reduced pressure. The residue was purified bychromatography over silica gel with dichloromethane as eluent to givethe product 212 as a clear oil (404 mg, 96%). MS FAB: 435 (M+H, 35%),457 (M+Na, 16%); Found: C, 71.76; H, 6.18; C₂₆H₂₆O₄S requires C, 71.86;H, 6.03%.(1S,3R,4R,7S)-7-Benzyloxy-1-benzyloxymethyl-3-phenylthio-2,5-dioxabicyclo[2.2.1]-heptane(212β). ¹H NMR (CDCl₃): δ 7.46-7.26 (15H, m, Bn, SPh), 5.35 (1H, s,H-1), 4.68-4.56 (4H, m, Bn), 4.31 (1H, s, H-2), 4.10 (1H, s, H-3), 4.09(1H, d, J 7.3 Hz, H-5′), 3.93 (1H, d, J 7.8 Hz, H-5′), 3.79 (2H, m,H-5); ¹³C NMR (CDCl₃): δ 138.03, 137.45, 133.42, 132.36, 129.19, 128.55,128.46, 128.05, 127.84, 127.83, 127.76 (Bn, SPh), 89.96 (C-1), 87.18(C-4), 79.71 (C-2), 79.40 (C-3), 73.64 (Bn), 73.23 (C-5′), 72.30 (Bn),66.31 (C-5).(1S,3S,4R,7S)-7-Benzyloxy-1-benzyloxymethyl-3-phenylthio-2,5-dioxabicyclo[2.2.1]heptane(212a). ¹H NMR (CDCl₃): δ 7.52-7.19 (15H, m, Bn, SPh), 5.52 (1H, s,H-1), 4.70-4.50 (4H, m, Bn), 4.41 (1H, s, H-2), 4.18 (1H, d, J 7.8 Hz,H-5′), 4.08 (1H, d, J 8.4 Hz, H-5′), 4.07 (1H, s, H-3), 3.78 (1H, d, J11.3 Hz, H-5), 3.72 (1H, d, J 11.5 Hz, H-5); ¹³C NMR (CDCl₃): δ 137.89,137.46, 135.29, 130.93, 129.13, 128.99, 128.57, 128.48, 127.81, 127.76,127.58, 126.95 (Bn, SPh), 91.87 (C-1), 88.59 (C-4), 80.07, 79.14 (C-2,C-3), 73.65, 73.40, 72.04 (Bn, C-5′), 65.62 (C-5).

Example 120

[0293] (3R)- and(3S)-(1S,4R,7S)-7-Benzyloxy-1-benzyloxymethyl-3-(thymin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane(36+213). Thymine (175 mg, 1.38 mmol) was stirred inhexamethyldisilazane (6.8 mL) at reflux and ammonium sulphate (5 mg) wasadded. After stirring for 16 h, the clear solution was cooled to 40° C.and the solvent was removed by distillation under reduced pressure. Tothe residue was added a solution of 212 (201 mg, 0.463 mmol) inanhydrous dichloromethane (4.6 mL) and 4 Å molecular sieves (180 mg).After stirring at room temperature for 10 min, NBS (107 mg, 0.602 mmol)was added and the mixture stirred for another 30 min. The reaction wasquenched with a saturated aqueous solution of sodium thiosulphate (25mL) and the resulting mixture was extracted with dichloromethane (2×50mL). The combined extract was dried (MgSO₄) and evaporated, and theresidue was purified on column chromatography over silica gel withdichloromethane:methanol (97:3) as eluent to give the product 36+213 andas an anomeric mixture (β:α˜1:2) (127 mg, 61%); ¹H NMR (CDCl₃): δ 7.49(d, J 0.9 Hz, H-6 β), 7.46 (d, J 1.0 Hz, H-6 α), 7.39-7.25 (m, Bn), 5.94(s, H-1′ α), 5.64 (s, H-1′ β), 4.71-4.50 (m, Bn, H-2′), 4.23 (s, H-3′α), 4.16 (d, J 8.6 Hz, H-5″ α), 4.09-3.78 (m, H-5′, H-5″, H-3′β), 1.94(d, J 0.9 Hz, CH₃ α), 1.62 (d, J 1.2 Hz, CH₃ β); MS FAB: 551 (M+H, 96%).

Example 121

[0294] (3R)- and(3S)-(1S,4R,7S)-7-Hydroxy-1-hydroxymethyl-3-(thymin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane(37+214). A solution of 36+213 (175 mg, 0.39 mmol) in ethanol (2.7 mL)was stirred at room temperature and 20% palladium hydroxide over carbon(50 mg) was added. The mixture was degassed several times with argon andplaced under a hydrogen atmosphere. After stirring for 18 h, the mixturewas purified on column chromatography over silica gel withdichloromethane:methanol (95:5) as eluent to give a mixture of 37 and214 (1:1.2) (26 mg, 25%); ¹H NMR (CD₃OD): δ 7.78 (d, J 1.3 Hz, H-6 α),7.73 (d, J 1.2 Hz, H-6 β), 5.88 (s, H-1′ α), 5.53 (s, H-1′ β), 4.38 (s,H-2′ α), 4.34 (s, H-3′ α), 4.26 (s, H-2′ β), 4.08-3.69 (m, H-5′, H-5″,H-3′β), 1.92 (d, J 1.2 Hz, CH₃ α), 1.88 (d, J 1.1 Hz, CH₃ β); ¹³C NMR(CD₃OD): δ 138.00 (C-6 α), 136.96 (C-6 β), 110.80 (C-5 β), 110.08 (C-5α), 92.49, 89.01 (C-4′, C-1′ α), 90.46, 88.37 (C-4′, C-1′ β), 80.89,74.27, 73.34 (C-2′, C-3′, C-5′ α), 80.59, 72.47, 70.39 (C-2′, C-3′, C-5′β), 59.29 (C-5″ α), 57.61 (C-5″ β), 12.52 (CH₃ α), 12.39 (CH₃ β); MS EI:270 (M+, 100%).

[0295] Preparation of LNA Phosphoramidites

Example 122

[0296] 4-N-Benzoyl-LNA-C[(1R,3R,4R,7S)-3-(4-N-benzoylcytosine-1-yl)-1-(hydroxymethyl)-7-hydroxy-2,5-dioxabicyclo{2.2.1} heptane]. LNA-C (formula Z) was taken in absolute ethanol andheated at reflux. To the refluxing solution, benzoic anhydride (2equivalents) was added and the reaction was followed by HPLC (Eluant:20% acetonitrile in 0.1M TEAA, pH 7.0, flow rate: 1 ml/min., NovapakC-18 analytical column). Additional anhydride was added at 0.5-2 hintervals till no more increase in product was observed by HPLC.Reaction mixture was concentrated on rotavap. Residue was repeatedlywashed with ether, filtered and dried to give an off white solid. Yield:45%.

[0297] General method for dimethoxytritylation of base protected LNAnucleosides (LNA-C^(Bz), LNA-T, LNA-G^(iBu), LNA-A^(Bz)). Base protectedLNA-nucleoside was coevaporated with pyridine (2×) and was stirred withdimethoxytrityl chloride (1.5 equivalents) in pyridine (˜10 ml/g ofnucleoside). The reaction was followed by HPLC (50% acetonitrile in 0.1MTEAA, pH 7.0, for 5 min., 50-100% acetonitrile in 10 min. and 100%acetonitrile for 5 min., flow rate: 1 ml/min., Novapak C-18 column).When >95% of the starting material had reacted, reaction mixture wascooled in ice. Reaction was quenched by addition of cold saturatedNaHCO₃ (˜15 ml×vol. of pyridine). The mixture was extracted withdichloromethane (3×half the vol. of sodium bicarbonate). Organicextractions were combined, dried over anhydrous sodium sulfate, filteredand concentrated on rotavap. Residue was dried in vacuo and purified bysilica gel chromatography using 0.5% pyridine and 0-2% methanol indichloromethane as eluant. Fractions containing pure products werecombined and concentrated on rotavap. Residue was coevaporated withanhydrous acetonitrile (3×) and dried in vacuo.

[0298] General method for phosphitylation of protected LNA nucleosides.Base protected dimethoxytrityl-LNA nucleoside was coevaporated withanhydrous dichloromethane (2×) and was taken in anhydrousdichloromethane (10 ml/g of nucleoside for A, G &T and ˜30 ml/g for C).To this bis(diisopropylamino)(2-cyanoethyl)phosphite (1.05-1.10equivalent), followed by tetrazole (0.95 equivalent) were added. Mixturewas stirred at room temperature and reaction was followed by HPLC (70%acetonitrile in 0.1M TEAA, pH 7, 2 min., 70-100% acetonitrile in 8 min.,and 100% acetonitrile in 5 min., flow rate: 1 ml/min., Novapak C-18column). Once the reaction had proceeded to >90% and no more increase inamidite formation was observed upon further stirring, the mixture wascooled in ice. It was diluted with dichloromethane (15-20 times theoriginal volume) and washed with cold saturated sodium bicarbonate (2×)followed by cold brine (1×). Organic layer was dried over anhydroussodium sulfate, filtered and concentrated on rotavap. Residue wascoevaporated with anhydrous acetonitrile (3×) and dried in vacuoovernight. HPLC purity ranged from 93-98%.

[0299] Preparation of LNA Nucleoside 5′-triphosphates

Example 123

[0300] Synthesis of LNA nucleoside 5′-triphosphates. (TetrahedronLetters 1988, 29 4525). In a 13×100 mm polypropylene tube, nucleosides37, 44, 51, 4-N-benzoylated 57A or 61B (93.8 μmol) was suspended in 1 mLpyridine (dried by CaH₂). The solution was evaporated in a speedvac,under high vacuum, to dryness. The residue was twice resuspended inacetonitrile (dried by CaH₂) and evaporated to dryness. The nucleosidewas suspended in 313 μL trimethyl phosphate (dried by 4 Å molecularsieves), to which 30.1 mg Proton Sponge™ (1.5 equivalents) were added.The mixture was sealed, vortexed, and cooled to 0° C. POCl₃ (9.8 μL, 1.1equivalent) was added with vortexing. The reaction was allowed toproceed at 0° C. for 2.5 hours. During this interval, 469 μmols sodiumpyrophosphate (5 equivalents) were dissolved in 5 mL water and passedthrough 5 mL Dow 50H⁺ ion exchange resin. When the effluent turnedacidic, it was collected in 220 μL tributylamine and evaporated to asyrup. The TBA pyrophosphate was coevaporated three times with dryacetonitrile. Finally, the dried pyrophosphate was dissolved in 1.3 mLDMF (4 Å sieves). After 2.5 hours reaction time, the TBA pyrophosphateand 130 μL tributylamine were added to the nucleoside solution withvigorous vortexing. After 1 minute, the reaction was quenched by adding3 mL 0.1 M triethylammonium acetate, pH 7.5. Assay by Mono Qchromatography showed 49% nucleoside 5′-triphosphate. The reactionmixture was diluted to 100 mL with water and adsorbed onto a Q Sepharoseion exchange column, washed with water, and eluted with a lineargradient of 0 to 700 mM NaCl in 5 mM sodium phosphate, pH 7.5. Fractionscontaining triphosphate were assayed by Mono Q ion exchangechromatography. Fractions containing triphosphate were pooled andconcentrated to the point of NaCl saturation. The product was desaltedon a C₁₈ cartridge. The triphospate was quantitated by UV spectroscopyand adjusted to 10 mM solution. Yields were 17-44%. LNA nucleosidesprepared by this method were, U, T, A, G, and C.

[0301] Preparation of LNA Modified Oligonucleotides

Example 124

[0302] Synthesis of oligonucleotides containing LNAs of formula V, X, Yand Z^(T), Z^(U), Z^(G), Z^(C), Z^(A), Z^(MeC). The bicyclic nucleoside3′-O-phosphoramidite analogues 8, 19, 30, 39, 46, 53, 57D, 61D, and 66as well as commercial 3′-O-phosphoramidites were used to synthesiseexample LNA oligonucleotides of the invention (0.2 to 5 μmol scale)containing one or more of the LNAs of types V, X, Y and Z^(T), Z^(U),Z^(G), Z^(C), Z^(A), and Z^(MeC). The purity and composition of thesynthesised LNA oligonucleotides was verified by capillary gelelectrophoresis, and/or HPLC and/or MALDI-MS. In general, satisfactorycoupling efficiencies were obtained for all the monomers. The bestcoupling efficiencies (˜95-100%) were obtained for LNAs 39, 46, 53, 57D,61D, and 66 (leading to LNA monomers of formula Z) giving verysatisfactory results when synthesising fully modified LNAoligonucleotides or when incorporating LNAs in otherwise unmodified DNAor RNA stands or LNAs into an all-phosphorothioate oligonucleotide. LNAoligonucleotides were dissolved in pure water and the concentrationdetermined as OD₂₆₀. Solubilities in all cases were excellent. For plainDNA/RNA synthesis and partially modified LNA oligomers, a standard CPGsupport or a polystyrene support, was used. For the synthesis of fullymodified LNA oligomers (e.g. 5′-d(GTGATATGC)-3′), a BioGenex UniversialCPG Support (BioGenex, U.S.A.) was used, or LNA derivatised supportswere used.)

Example 125

[0303] Synthesis of phosphorothioate LNA oligonucleotides. Theall-phosphorothioate LNA (Table 7) was synthesised on an automated DNAsynthesiser using similar conditions as those described earlier (Example124). Beaucages' reagent was used as sulphurising agent. The stepwisecoupling yields were >98%. After completion of the syntheses,deprotection and cleavage from the solid support was effected usingconcentrated ammonia (55° C., 14 h).

Example 126

[0304] Synthesis of 2′-Thio-LNA oligonucleotides. The 2′-thio-LNAoligonucleotides (containing monomer U^(S) (formula Z (thio-variant) ofFIG. 2), FIG. 37, Table 8) were synthesised on an automated DNAsynthesiser using standard conditions (Example 124). The step-wisecoupling yield for amidite 76F was approximately 85% (12 min couplings;improved purity of amidite 76F is expected to result in increasedcoupling yield). After completion of the syntheses, deprotection andcleavage from the solid support was effected using concentrated ammonia(55° C., 8 h).

Example 127

[0305] Synthesis of 2′-Amino-LNA oligonucleotides. By procedures similarto those described in Example 126, 2′-Amino-LNA oligonucleotides(containing monomer T^(NH) and monomer T_(NMe) (formula Z (aminovariants) of FIG. 2), FIGS. 35 and 36) was efficiently obtained on anautomated DNA synthesiser using amidites 74A and 74F (≧98% stepwisecoupling yields).

Example 128

[0306] Fluorescein-labeling of LNA oligomers. LNA oligomers (formula Zof FIG. 2) AL16 (5′-d(TGTGTGAAATTGTTAT)-3′; LNA nucleotides in bold) andAL17 (5′-d(ATAAAGTGTAAAG)-3′; LNA nucleotides in bold) were succesfullylabeled with fluorescein using the FluoroAmp T4 Kinase GreenOligonucleotide Labeling System as described by the manufacturer(Promega). Briefly, 16 mmol of either LNA-oligomer AL16 or AL17 was5′-thiophosphate labelled in a 50 μl reaction buffer containing T4kinase and γ-S-ATP. The reactions were incubated for 2 h at 37° C. Thethio-phosphorylated LNA oligos were precipitated by the addition of 5 μlof oligonucleotide precipitant (Promega) and 165 μl of ice cold (−20°C.) 95% ethanol. After centrifugation the pellets were washed once with500 μl of ice cold (−20° C.) 70% ethanol and redissolved in 25 μl ofPBSE buffer. Freshly prepared 5-maleimide-fluorescein solution (50 μg in5 μl DMSO) were added to the thiophosphorylated LNA oligos and thereaction mixtures incubated at 68° C. for 30 min. Additional5-maleimide-fluorescein (50 μg in 5 μl DMSO) were added to each LNAoligo and the reaction mixtures incubated for an additional 60 min.After incubation 10 μl of oligonucleotide precipitant was added to eachreaction mixture followed by 180 μl ice-cold (−20° C.) and 100 μlN,N-dimethylformamide. The fluorescein labeled LNA oligos were isolatedby centrifugation followed by aspiration of the supernatant. Thefluorescein labelled LNA-oligomers were purified by reversed-phase HPLCas follows: column Delta-Pack C-18, 300A, 0.4×30 cm; eluent 0-50%acetonitrile in 0.04 M triethylammonium buffer (pH 7.0); flow rate 1.5ml/min. The fractions containing LNA-oligos were pooled and evaporatedunder reduced pressure (oil pump and speed-vac system) during 12 h.

[0307] Hybridisation Data

Example 129

[0308] Thermostability of oligonucleotides containing monomers offormula V, X, Y and Z^(T), Z^(U), Z^(G), Z^(C), Z^(A), Z^(MeC). Thethermostability of the LNA modified oligonucleotides were determinedspectrophotometrically using a spectrophotometer equipped with athermoregulated Peltier element. Hybridisation mixtures of 1 ml wereprepared containing either of 3 different buffers (10 mM Na₂HPO₄, pH7.0, 100 mM NaCl, 0.1 mM EDTA; 10 mM Na₂HPO₄ pH 7.0, 0.1 mM EDTA; 3Mtetrametylammoniumchlorid (TMAC), 10 mM Na₂HPO₄, pH 7.0, 0.1 mM EDTA)and equimolar (1 μM or 1.5 μM) amounts of the different LNA modifiedoligonucleotides and their complementary or mismatched DNA or RNAoligonucleotides. Identical hybridisation mixtures using the unmodifiedoligonucleotides were prepared as references. The T_(m)'s were obtainedas the first derivative of the melting curves. Tables 1-4 summarise theresults (LNAs are marked with bold). FIG. 2 illustrates the monomericLNAs used. The nomenclature V, X, Y and Z^(T), Z^(U), Z^(G), Z^(C),Z^(A), Z^(MeC) refer to structures V, X, Y and Z of FIG. 2. In thetables, the nucleobases of the LNA monomers are indicated. Furthermore,for the thio and amino variants of the LNA structure Z of the last twotables, the nomenclature used is, e.g., Z^(TS) and Z^(TNH),respectively.

[0309] LNAs containing structure Z were particularly thoroughly examined(see Table 1). When three Z^(T) residues were incorporated into anoligonucleotide of mixed sequence the T_(m)'s obtained in NaCl bufferwith both complementary DNA (10) and RNA (16) oligonucleotides weresubstantially higher (RNA: roughly 7° C. and DNA: roughly 5° C. permodification) than the T_(m) of the corresponding duplexes withunmodified oligonucleotides (1 and 8). Similar results were obtainedwith LNAs containing two Z^(T) residues and either one Z^(G) (21 and24B) or Z^(U) (25), Z^(C) (69), Z^(MeC) (65), and Z^(A) (58) residues.When mismatches were introduced into the target RNA or DNAoligonucleotides the T_(m) of the LNA modified oligonucleotides in allcases dropped significantly (1-15A and 17; 18-20 and 22-24A; 26-31; 57and 59-60; 63-64 and 66, and 67), unambiguously demonstrating that theLNA modified oligonucleotides hybridise to their target sequencesobeying the Watson-Crick hydrogen bonding rules. In all cases the dropin T_(m) of the LNA modified oligonucleotides upon introduction ofmismatches was equal to or greater than that of the correspondingunmodified oligonucleotides (2-7 and 9; 33-38), showing that the LNAmodified oligonucleotide are at least as specific as their naturalcounterparts. A lowering of the ionic strength of the hybridisationbuffer (from 10 mM Na₂HPO₄, pH 7.0, 100 mM NaCl, 0.1 mM EDTA to 10 mMNa₂HPO₄ pH 7.0, 0.1 mM EDTA) lowers the T_(m) of the LNA modifiedoligonucleotides for their complementary DNA oligos (40,41) or RNAoligonucleotides (40A, 41A). A similar effect is observed with theunmodified oligonucleotides and its complementary DNA oligo (39) or RNAoligo (39A).

[0310] Addition of 3M tetrametylammoniumchlorid (TMAC) to thehybridisation buffer significantly increases the T_(m) of the LNAmodified oligonucleotide for their complementary DNA oligos (10, 21,25). Moreover, TMAC levels out the diffeneces in the T_(m)'s of thedifferent oligonucleotides which is observed in the NaCl buffer (lowestT_(m) in the NaCl buffer 44° C. and highest 49° C. as opposed to 56° C.and 57° C. in TMAC). Introduction of mismatches substantially decreasesthe T_(m) of the LNA modified oligonucleotides for their DNA targets(11-13, 18-20, and 26-28). A similar picture emerges with the unmodifiedreference oligonucleotides (1-4 and 32-35)

[0311] The data with the low salt buffer shows that LNA modifiedoligonucleotides exhibit a sensitivity to the ionic strength of thehybridisation buffer similar to normal oligonucleotides. From the T_(m)data with the TMAC buffer we infer that TMAC exhibits a T_(m) equalisingeffect on LNA modified oligonucleotides similar to the effect observedwith normal DNA oligonucleotides. LNA modified oligonucleotides retaintheir exquisite specificity in both hybridisation buffers.

[0312] The fully modified LNA oligonucleotide containing all fourmonomers (71 and 75), the almost fully modified LNA oligonucleotide(except for a 3′-terminal DNA nucleoside) containing both Z^(G) andZ^(T) (41 and 41A) and the partly modified oligonucleotide containing acentral block of Z^(T) and Z^(G) (40 and 40A) also exhibit substantiallyincreased affinity compared to the unmodified control oligonucleotide(39 and 39A; 1 and 8). This shows that LNAs of formula Z are very usefulin the production of both fully and partly modified oligomers. We notethat the almost fully modified oligomer (41 and 41A) exhibits anunprecedented high affinity for both complementory RNA (>93° C.) and DNA(83° C.). A similar extreme affinity (for both RNA and DNA) was observedwith the almost fully modified LNA oligomer containing exclusively Z^(T)(Table 1: 52 and 53) and the fully modified LNA oligomer (71 and 75).The affinity of the partly modified poly-T oligonucleotide depended onthe positions and the number of Z^(T) monomers incorporated (44-51).Whereas the T_(m)'s with RNA targets (45, 47, 49 and 51) in all caseswere higher than the corresponding unmodified oligonucleotides (43) onegave a lower T_(m) with the DNA target (46). Since mixed sequenceoligonucleotide containing 3 Z^(T) residues exhibited a substantiallyincreased affinity for their DNA (10) and RNA target (16) compared tothe unmodified reference oligonucleotides (1 and 8) this suggests thatother binding motifs than Watson-Crick (such as for example theHoogsteen binding motif) are open to poly-T oligonucleotides and thatthese binding motifs are somewhat sensitive to the precise architectureof the modified oligonucleotide. In all cases introduction of singlebase mismatches into the complex between the fully Z^(T) modified poly-Toligonucleotide and a DNA target (54-56) resulted in a significant dropin T_(m).

[0313] Oligonucleotides containing either LNAs of structures V (Table2), X (Table 3) and Y (Table 4) were analysed in the context of fullyand partly modified poly-T sequences. The fully modifiedoligonucleotides of structure V and Y exhibited an increase in T_(m)(albeit much lower than the Z^(T) modified oligonucleotides) with bothRNA (Table 2, 14 and Table 4, 14) and DNA targets (Table 2, 13, andTable 4, 13) compared to the unmodifed oligonucleotides (Table 1, 42 and43). The partly modified oligonucleotides containing monomers ofstructure V and Y behaved similarly to partly modified oligonucleotidescontaining Z^(T) and probably this is due to the homopolymer nature ofthe sequence as outlined above. Oligonucleotides containing X^(T) in allcases exhibited a much reduced T_(m) compared to the reference DNAoligonucleotides.

Example 130

[0314] A fully modified LNA oligonucleotide form stable hybrids withcomplementary DNA in both the anti-parallel and the parallelorientation. A full modified LNA oligonucleotide was hybridised to itscomplementary DNA in both the anti-parallel and the parallelorientation. Hybridisation solutions (1 mL) contained 10 mM Na₂HPO₄ (pH7), 100 mM NaCl and 0.1 mM EDTA and 1 μM of each of the twoolignucleotides. As shown in Table 1 both the anti-parallel (71) and theparallel binding orientation (77) produces stable duplexes. Theanti-parallel is clearly the most stable of the two. However, even theparallel duplex is significantly more stable than the correspondinganti-parallel duplex of the unmodified DNA oligonucleotides (Table 1,1).

Example 131

[0315] LNA monomers can be used to increase the affinity of RNAoligomers for their complementary nucleic acids. The thermostability ofcomplexes between a 9-mer RNA oligonucleotide containing 3 LNA-Tmonomers (Z^(T)) and the complementary DNA or RNA oligonucleotides weremeasured spectrophotometrically. Hybridisation solutions (1 ml)containing 10 mM Na₂HPO₄, pH 7.0, 100 mM NaCl, 0.1 mM EDTA and 1 μM ofeach of the two oligonucleotides. Identical hybridisation mixtures usingthe unmodified RNA oligonucleotides were measured as references. Asshown in Table 5 the LNA modified RNA oligonucleotide hybridises to bothits complementary DNA (1) and RNA (3) oligonucleotide. As previouslyobserved for LNA modified DNA oligonucleotides, the binding affinity ofthe LNA modified RNA oligonucleotide is strongest to the RNA complement(3). In both cases the affinity of the LNA modified RNA oligonucleotideis substantially higher than that of the unmodified controls (2 and 4).Table 5 also shows that the specificity towards both DNA and RNA targetsare retained in LNA modified RNA oligonucleotides.

Example 132

[0316] LNA-LNA base pairing. RNA or DNA oligonucleotides containingthree Z^(T) LNA monomers or an oligonucleotide composed entirely of LNAZ monomers were hybridised to complementary unmodified DNAoligonucleotides or DNA oligonucleotides containing three Z^(A) LNAmonomers and the T_(m) of the hybrids were measuredspectrophotometrically. Hybridisation solutions (1 ml) contained 10 mMNa₂HPO₄, pH 7.0, 100 mM NaCl and 0.1 mM EDTA and 1 μM of each of the twooligonucleotides. As shown in Table 6 all the LNA modifiedoligonucleotides hybridises to the complementary, unmodified DNAoligonucleotides (2 and 3) as well as the complementary LNA modifiedoligonucleotides (4, 5 and 6). As observed previously the presence ofLNA monomers in one strand of a hybrid (2 and 3) increases the T_(M)significantly compared to the unmodified control hybrid (1). Thepresence of LNA-LNA base pairs in the hybrid increases the T_(M) evenfurther (4 and 5) Moreover, a highly stable hybrid can be formed betweena fully modified LNA oligonucleotide and a partly LNA-Z^(A) modified DNAoligonucleotide (6). This constitutes the first example of LNA-LNA basepairs in a hybrid.

Example 133

[0317] An LNA all-phosphoromonothioate oligonucleotide displayrelatively less decreased thermostability towards complementary DNA andRNA than the corresponsing all-phosphorothioate DNA oligonucleotide. Thethermostability of an all-phosphoromonothioate DNA oligonucleotidecontaining three Z^(T) LNA monomers (LNA oligonucleotide) and thecorresponding all-phosphoromonothioate reference DNA oligonucleotidetowards complementary DNA and RNA was evaluated under the sameconditions as described in Example 132, however without EDTA (Table 7).It was observed that the LNA all-phosphoromonothioate oligonucleotidecontaining three LNA Z^(T) monomers displayed only weakly decreasedthermostability (Table 7, 3 and 4) when compared to the correspondingreference LNA oligonucleotide (Table 1, 10 and 16). The correspondingall-phosphoromonothioate DNA oligonucleotide (Table 7, 1 and 2)displayed significantly decreased thermostability when compared to thecorresponding reference DNA oligonucleotide (Table 1, 1 and 8). This hasimportant possible implications on the use of all- or partiallyphosphoromonothioate LNA oligonucleotides in antisense and othertherapeutic applications. Thus, the compatibility of LNA monomers andunmodified monomers in an phosphoromonothioate oligonucleotide has beendemonstrated. It can be anticipated that such constructs will displayboth Rnase H activity and nuclease resistance in addition to the LNAenhanced hybridisation characteristics.

Example 134

[0318] 2′-Thio-LNA display nucleic acid recognition propertiescomparable with those of LNA (Monomer Z). The hybridisation conditionswere as described in Example 132, however without EDTA. The results forthe 2′-thio-LNAs (Table 8) clearly indicate a positive effect on thethermal stability of duplexes towards both DNA and RNA by theintroduction of 2′-thio-LNA monomer U^(S) (The monomers correspond toformula Z of FIG. 2 where the methyleneoxy bridge has been substitutedwith a methylenethio bridge). This effect (ΔT_(m)˜+5° C./modificationtowards DNA; ΔT_(m)˜+8° C./modification towards RNA) is comparable withthat observed for parent LNA. The picture is complicated by thesimultaneous introduction of two modifications (the 2′-thiofunctionality and uracil instead of thymine). However, as we haveearlier observed identical melting temperatures for the LNA thymine anduracil monomers, and as the references containing 2′-deoxyuridineinstead of thymidine, if anything, would be expected to display lowerT_(m) values, the comparison is relevant.

Example 135

[0319] 2′-Amino-LNA (Monomer Z^(TNH)) and 2′-Methylamino-LNA (MonomerZ^(TNMe)) display nucleic acid recognition properties comparable withthose of parent LNA (Monomer Z). The hybridisation conditions were asdescribed in Example 132, however without EDTA. The melting results forthe 2′-amino-LNAs (Table 9) clearly indicate a positive effect on thethermal stability of duplexes towards DNA and RNA by introduction ofeither 2′-amino-LNA monomers T^(NH) or T^(NMe) (The monomers correspondto formula Z of FIG. 2 where the methyleneoxy bridge has beensubstituted with a methyleneamino bridge or methylene-(N-methyl)aminobridge, respectively). This effect (ΔT_(m)˜+3° C./modification towardsDNA and ΔT_(m)˜+6 to +8° C./modification towards RNA) is comparable tothat of parent LNA. It is notheworthy, that the increased thermalaffinity is also observed with an oligo composed of a mixture of2′-alkylamino-LNA monomers and nonalkylated 2′-amino-LNA monomers.

[0320] LNA and LNA Modified Oligonucleotides as a Substrates for Enzymes

Example 136

[0321] 3′-Exonucleolytic stability of oligomers 5′-V^(T) ₁₃T and 5′Z^(T) ₁₃T. A solution of oligonucleotides (0.2 OD) in 2 ml of thefollowing buffer (0.1 M Tris-HCl, pH 8.6, 0.1 M NaCl, 14 mM MgCl₂) wasdigested at 25° C. with 1.2 U SVPDE (snake venom phosphodiesterase).During digestion, the increase in absorbance at 260 nm was followed.Whereas the unmodified control T₁₄ was fully degraded after 10 min ofdegradation, 5-Z^(T) ₁₃T and 5′-V_(T) ₁₃T remained intact for 60 min.

Example 137

[0322] LNA modified oligos as substrates for T4 polynucleotide kinase.20 pmoles of each primer (FP2: 5′-GGTGGTTTGTTTG-3′; DNA probe), (AL2:5′-GGTGGTTTGTTTG-3′, LNA nucleosides in bold) and (AL3:5′-GGTGGTTTGTTTG-3′, LNA nucleosides in bold) was mixed with T4polynucleotide Kinase (5 Units; New England Biolabs) and 6 μlγ-^(32P)ATP (3000 Ci/mmol, Amersham) in a buffer containing 70 mMTris-HCl (pH 7.6), 10 mM MgCl₂, 5 mM dithiotretiol (final volume 20 pi).The samples were incubated 40 min at 37° C. and afterwards heated to 65°C. for 5 min. To each of the reactions were added 2 μl of tRNA (1μg/μl), 29 μl of a 3M ammonium acetate and 100 μl of ethanol. Thereactions were incubated at −20° C. for 30 min. and the labelled oligoswere precipitated by centrifugation at 15000 g for 30 min. The pelletwas resuspended in 20 μl H₂O. The samples (1 μp) were mixed with aloading buffer (formamide (pH 8.0), 0.1% xylene cyanol FF, 0.1%bromophenol blue and 10 mM EDTA) and electrophoresed on a denaturingpolyacrylamide gel (16% acrylamide/bisacrylamide solution, 7 M urea,1×TBE and 0.1 mM EDTA) in a TBE running buffer (90 mM Tris-HCl (pH 8.3),90 mM boric acid and 2.5 mM disodium EDTA-2H₂O). The gel was dried on agel dryer (BioRad model 583) and autoradiographed to a X-ray film(CL-XPosure film, Pierce 34075) for 20 min. The result is shown in FIG.6 (FP2: lane 1 and 2; AL2: lane 3 and 4; AL3: lane 5 and 6). Threeconclusions can be drawn on the basis of this experiment. Firstly, itcan be concluded that partly and fully LNA modified oligos are excellentmimics of natural nucleic acid in their ability to act as substrate fora nucleic acid specific enzyme like polynucleotide kinase. Secondly, itcan be concluded that LNA modified oligos can be efficientlyprecipitated by procedures normally employed to precipitate standardnucleic acids. In fact, the relative signal intencities of theunmodified (lane 1,2), partly (lane 3,4) and fully modified oligos (lane5,6) in the autoradiogram suggests that the more LNA nucleosides astandard DNA oligo contains the more efficiently it can be precipitatedby salt/alcohol procedures. Thirdly, the similar positions of the signalin the autoradiogram of the unmodified, partly and fully modified oligosshows that incorporation of LNA nucleosides into a DNA oligo does notalter its electrophoretic mobility in polyacrylamide gels.

Example 138

[0323] 3′-End labelling of LNA-containing oligonucleotides with terminaldeoxynucleotidyl transferase. Oligonucleotides containing LNA monomerswere 3′end-labelled using the enzyme terminal deoxynucleotidyltransferase. The sequence and extent of LNA modification were as follows(where LNA monomers are in bold):

[0324] Control 5′ GGT GGT TTG TTT G 3′

[0325] (1) 5′ GGT GGT TTG TTT G 3′

[0326] (2) 5′ GGT GGT TTG TTT G 3′

[0327] (3) 5′ GGT GGT TTG TTT G 3′

[0328] Oligonucleotide (50 pmol) was incubated with 250 μCi [α³²P]ddATP(3000 Ci/mmol) and 100 Units terminal deoxynucleotidyl transferase in250 μl 100 mM cacodylate buffer pH 7.2, 2 mM CoCl₂ and 0.2 mM2-mercaptoethanol at 37° C. for 2 hours. The reaction was then stoppedby adding formamide loading buffer and heating to 100° C. for 5 minbefore placing on ice. Samples (0.2 pmol) were run on a 19% acrylamidegel containing 7M urea and the percentage incorporation of radioactivityinto the oligonucleotide bands was quantified by means of aphosphorimager (Molecular Dynamics). The results show incorporation ofradioactivity in all cases, including the oligonucleotide with a highLNA content: Control 94.9%, (1) 39.7%, (2) 83.7%, (3) 31.7%. We concludethat LNA modified oligos are substrates for the TdT enzyme.

Example 139

[0329] The ability of terminal deoxynucleotidyl transferase (TdT) totail LNA modified oligonucleotides depends on the design of theoligomer. The following 15 mer primers and a mixture of 8 to 32 baseoligonucleotide markers were 5′ end labelled with [γ³³P] ATP and T4polynucleotide kinase (where LNA monomers are in bold):

[0330] P1 5′-TGC ATG TGC TGG AGA-3′

[0331] P2 5′-GC ATG TGC TGG AGA T-3′

[0332] PZ1 5′-TGC ATG TGC TGG AGA-3′

[0333] PZ2 5′-GC ATG TGC TGG AGA T-3′

[0334] Reactions were boiled for 5 min after labelling to remove any PNKactivity. Four picomoles of each labelled primer, 25 U terminaldeoxynucleotidyl transferase and 16 μM dATP were incubated in 25 μl 100mM cacodylate buffer pH 7.2, 2 mM CoCl₂ and 0.2 mM 2-mercaptoethanol for90 min at 37° C. The reactions were stopped by the addition of formamidestop solution and the reaction products run on a 19% polyacrylamide 7 Murea gel with the labelled markers. Autoradiography using Biomax filmwas carried out on the dry gel. As shown in FIG. 22, P1 (lane 2), P2(lane 4) and PZ1 (lane 3) all gave a tail estimated at greater than 70bases long on the basis of the 8-32 base marker (lanes 1 and 6). PrimerPZ2 (lane 5) was not extended under these reaction conditions. Weconclude that the TdT enzyme will tolerate LNA monomers within theoligonucleotide, but not at the extreme 3′ end.

Example 140

[0335] LNA-thymidine-5′-triphosphate (LNA-TTP) as a substrate forterminal deoxynucleotidyl transferase (TdT). In order to test theability of the triphosphate of LNA-TTP (Example 123) to be accepted byterminal deoxynucleotidyl transferase as a substrate, an oligonucleotidetailing reaction was performed. A 15 mer primer (sequence: 5′-TGC ATGTGC TGG AGA-3′) and a mixture of 8 to 32 base oligonucleotide markerswere 5′ end labelled with [γ³³P] ATP and T4 polynucleotide kinase.Reactions were boiled for 5 min after labelling to remove any PNKactivity. Four picomoles of the labelled primer, 25 U terminaldeoxynucleotidyl transferase and 32, 64 or 128 μM dTTP or LNA-TTP wereincubated in 25 μl 100 mM cacodylate buffer pH 7.2, 2 mM CoCl₂ and 0.2mM 2-mercaptoethanol for 90 min at 37° C. The reactions were stopped bythe addition of formamide stop solution and the reaction products run ona 19% polyacrylamide 7M urea gel with the labelled markers.Autoradiography using Biomax film was carried out on the dry gel. Asshown in FIG. 10, reactions with either 32 μM dTTP (lane B), 64 μM dTTP(lane C) or 128 μM dTTP (lane D) all produced tailed oligonucleotideswhich on the basis on the 8-32 oligonucleotide marker (outermost leftand rigth lanes) were estimated at greater than 100 nucleotides. TheLNA-TTP reactions (32 μM dTTP (lane E), 64 μM dTTP (lane F) or 128 μMdTTP (lane G)) all resulted in the primer being extended by one base and50% of this being extended by a further base. This result is verysimilar to that obtained with ribonucleotides and TdT. We conclude thatLNA derived triphosphates can be recognised and incorporated into a DNAoligonucleotide by the TdT enzyme. This latter finding that LNA-TTP canbind to the polymerase underscores the possibility of successfully usingLNA-monomer derivatives as nucleoside drugs.

Example 141

[0336] Exonuclease free Kienow fragment DNA polymerase I can incorporateLNA Adenosine, Cytosine, Guanosine and Uridine-5′-triphosphates (LNAATP, LNA CTP, LNA GTP, LNA UTP) into a DNA strand. A primer extensionassay was used to evaluate the LNA NTP's (see Example 123),ribonucleotides, as substrates for exonuclease free Klenow fragment DNApolymerase I (EFK). The assay used a ³³P 5′ end labelled 15 mer primerhybridised to one of four different 24 mer templates. The sequences ofthe primer and templates are (LNA monomer in bold):

[0337] Primer 5′ TGCATGTGCTGGAGA 3′

[0338] Template 1 3′ ACGTACACGACCTCTACCTTGCTA 5′

[0339] Template 2 3′ ACGTACACGACCTCTCTTGATCAG 5′

[0340] Template 3 3′ ACGTACACGACCTCTTGGCTAGTC 5′

[0341] Template 4 3′ ACGTACACGACCTCTGAACTAGTC 5′

[0342] One picomole ³³P labelled primer was hybridised to 2 picomoles oftemplate in ×2 Klenow buffer. To this was added either 4 μM dNTPαS or500 μM LNA NTP or a mixture of 4 μM dNTPαS and 500 μM LNA NTP. Two unitsof EFK DNA polymerase was added to each reaction. 2 mU inorganicpyrophosphatase was added to each of the reactions. Primer plus templateplus enzyme controls were also carried out. All reactions were carriedout in a total volume of 20 W. The reactions were incubated at 37° C.for 3 min. Reactions were then stopped by the addition of 10 μlformamide EDTA stop solution. Reaction products were separated on a 19%polyacrylamide 7M urea gel and the product fragments sized by comparisonwith a ³³P labelled 8 to 32 base oligonucleotide ladder after exposureto Kodak Biomax autoradiography film.

[0343]FIG. 20 shows the result with LNA-UTP using template 1. The tracks(1-12) correspond to the following reactions: Incorporation of LNA UTPby EFK. Lane 1—Primer, template and enzyme. Lane 2—plus dTTPαS. Lane3—plus LNA UTP. Lane 4—plus dTTPαS and dGTPαS. Lane 5—plus LNA UTP anddGTPαS. Lane 6—plus dATPαS, dGTPαS and dTTPαS. Lane 7—plus LNA UTP,dCTPαS, dGTPαS and dTTPαS. Lane 8—plus dGTPαS. Lane 9—plus dCTPαS,dGTPαS and dTTPαS. Lane 10—plus LNA UTP, dATPαS, dCTPαS and dGTPαS. Lane11—plus dATPαS, dCTPαS and dGTPαS. Lane 12—all 4 dNTPαS. The laneseither side show the 8-32 base oligonucleotide markers used for sizingthe products.

[0344] As is evident from FIG. 20, LNA UTP is specifically incorporatedas a “T”. Further extension from an LNA UTP terminated 3′ end withdNTPαS is very slow.

[0345]FIG. 21 shows the result with LNA-ATP, LNA CTP, and LNA GTP usingtemplate 2-4. The tracks (1-21) correspond to the following reactions:Lanes 1, 7, 13 and 17—primer, template and enzyme. Lane 2—plus dGTPαS.Lane 3—plus dATPαS and dGTPαS. Lane 4—plus LNA GTP. Lane 5—plus dGTPαSand LNA ATP. Lane 6—plus LNA ATP and LNA GTP. Lane 8—plus dATPαS. Lane9—plus dATPαS and dCTPαS. Lane 10—plus LNA ATP. Lane 11—plus dCTPαS andLNA ATP. Lane 12—plus dATPαS and LNA CTP. Lane 14—plus dTTPαS. Lane15—plus dGTPαS and dTTPαS. Lane 16—plus dTTPαS and LNA GTP. Lane 18—plusdCTPαS. Lane 19—plus dCTPαS and dTTPαS. Lane 20—plus LNA CTP. Lane21—dTTPαS and LNA CTP. The lanes either side show the 8-32 baseoligonucleotide markers used for sizing the products.

[0346] The experiments using template 2 (track 1-6), show that LNA GTPis able to produce the +1 product with efficient extension of the primer(track 4). The addition of dGTPαS and LNA ATP results in mainly the +2product (track 5). This is from the incorporation of dGTPαS to give the+1 product followed by extension with LNA ATP. There is evidence of asmall amount of +3 product from the consecutive incorporation of LNAATP. The experiments using Template 3 (tracks 7-12) show that LNA ATP isefficiently incorporated to give the +1 product (track 10). Extension ofthis product with dCTPαS is slow (track 11). The addition of dATPαS andLNA CTP results in the +2 and +3 products (track 12). The absence of anysignificant +1 product shows that the addition of the first LNA CTP isefficient, but that the addition of the second LNA CTP is slow. Theresults from experiments on Templates 1 (tracks 13-16) and 4 (tracks17-21) show similar trends to those on the other templates. LNA CTP isefficiently incorporated to give the +1 product on Template 4 (track20). Extension of this product by dTTPαS is again slow (track 21). Theaddition of LNA GTP and dTTPαS to reactions on Template 1 results in the+2 product (track 16). Again this shows that the addition of a singleLNA triphosphate is quite efficient, but that the addition ofconsecutive LNA triphosphates is slow.

Example 142

[0347] LNA monomers can be used to enhance the resistance of anoligonucleotide to digestion by exonuclease III. In order to test theresistance of the LNA containing oligonucleotides to Exonuclease IIIdegradation the following reaction was performed. The following 15 merprimers and 8 to 32 base oligonucleotide markers were 5′ end labelledwith [γ³³P] ATP and T4 polynucleotide kinase (LNA monomer in bold):

[0348] P2 5′-GC ATG TGC TGG AGA T-3′

[0349] PZ2 5′-GC ATG TGC TGG AGA T-3′

[0350] Reactions were boiled for 5 min after labelling to remove any PNKactivity. 8 picomoles of each primer was hybridised to 25 pmolesTemplate (sequence: 3′-ACG TAC ACG ACC TCT ACC TTG CTA-5′) in ×2 Klenowbuffer. 10 Units of Exonuclease III was added to each of the reactions.Controls were also set up which had 1 μl water added in place of theenzyme. The reactions were incubated at 37° C. for 5 min. The reactionswere stopped by the addition of 10 μl formamide/EDTA stop solution. Thereactions were heated at 95° C. for 3 min before loading onto a 19%polyacrylamide 7M urea gel. The gel was fixed in 10% acetic acid/i 0%methanol before transferring to 3 MM paper and drying. The dried gel wasexposed to a phosphor screen for 3 hours. The phosphor screen wasanalysed on the Molecular Dynamics Storm 860 instrument using ImageQuantsoftware. The phosphor screen analysis showed that in the absence of theenzyme the P2 full length band was 99% of the signal and PZ2 full lengthband was 96% of the signal. In the presence of the enzyme only 20% ofthe P2 full length product was left after the 5 minute incubation.However, 62% of the full length PZ2 product remained after the sametreatment. This shows that a single LNA monomer at the 3′ end of anoligonucleotide can enhance the resistance to degradation by exonucleaseIII. PCR applications

Example 143

[0351] LNA monomers can be used to significantly increase theperformance of biotinylated-DNA oligos in the sequence specific captureof PCR amplicons in a MTP format. Two DIG labelled amplicons from pUC19were generated by PCR amplification as follows:

[0352] PCR Reaction Mixture for Amplicon 1

[0353] 1 μl pUC19 (1 ng/μl),

[0354] 1 μl reverse primer (5′-AACAGCTATGACCATG-3′) (20 μM),

[0355] 1 μl forward primer (5′-GTAAAACGACGGCCAGT-3′) (20 μM),

[0356] 10 μl dUTP-mix (2 mM dATP, 2 mM dCTP, 2 mM dGTP and 6 mM dUTP),

[0357] 1.5 μl DIG-11-dUTP (1 mM)

[0358] 10 μl 10×Taq buffer (Boehringer Mannheim inci MgCl₂)

[0359] 1 μl Taq polymerase (Boehringer Mannheim) 5 U/μl

[0360] H₂O ad 100 μl

[0361] PCR Reaction Mixture for Amplicon 2

[0362] 1 μl pUC19 (1 ng/μl),

[0363] 0.4 μl primer 3 (5′-GATAGGTGCCTCACTGAT-3′) (50 μM),

[0364] 0.4 μl primer 4 (5′-GTCGTTCGCTCCAAGCTG-3′) (50 μM),

[0365] 10 μl dUTP-mix (2 mM dATP, 2 mM dCTP, 2 mM dGTP and 6 mM dUTP),

[0366] 1.5 μl DIG-11-dUTP (1 mM)

[0367] 10 μl 10×Taq buffer (Boehringer Mannheim incl MgCl₂)

[0368] 1 μl Taq polymerase (Boehringer Mannheim) 5 U/μl

[0369] H₂O ad 100 μl

[0370] PCR reaction: (Cycler: Perkin Elmer 9600) 94° C. 5 min; addpolymerase; 94° C. 1 min, 45° C. 1 min, 70° C. 2 min (29 cycles) 72° C.10 min.

[0371] 10 μl from each PCR reaction was analysed on a standard agarosegel and the expected fragments of approximately 100 bp and 500 bp wereobserved.

[0372] 10 μl of DIG-labelled amplicon 1 or amplicon 2 was mixed with 5pmol of 5′ biotinylated capture probe in 1×SSC (0.15 M NaCl, 15 mMcitrate, pH 7.0) in a total volume of 450 μl. The following captureprobes were used: B-DNA1 (biotin-ATGCCTGCAGGTCGAC-3′; DNA probe specificfor amplicon 1), B-DNA2 (biotin-GGTGGTTTGTTTG-3′; DNA probe specific foramplicon 2) and B-LNA2 (biotin-GGTGGTTTGTTTG-3′, LNA nucleosides inbold; LNA probe specific for amplicon 2). Reactions were heated to 95°C. for 5 min in order to denature amplicons and allowed to cool at 25°C. for 15 min to facilitate hybridisation between the probe and thetarget amplicon strand. After hybridisation 190 μl of each reaction weretransferred to a streptavidin coated micro plate (Pierce, cat. no.1 5124) and incubated for one hour at 37° C. After washing the plate withphosphate buffered saline (PBST, 0.15 M Na⁺, pH 7.2, 0.05% Tween 20,3×300 μl), 20 μl of peroxidase labelled anti-DIG antibodies were added(Boehringer Mannheim, diluted 1:1000 in PBST). Plates were incubated for30 min at 37° C. and washed (PBST, 3×300 μl). Wells were assayed forperoxidase activity by adding 100 pi of substrate solution (0.1 Mcitrate-phosphate buffer pH 5.0, 0.66 mg/ml ortho-pheylenediaminedihydrochloride, 0.012% H₂O₂). The reaction was stopped after 8 min byadding 100 μl H₂SO₄ (0.5 M) and the absorbance at 492 nm was read in amicro plate reader. As shown in FIG. 3, the unmodified bio-DNAs captureprobes (B-DNA1 and B-DNA2) both behave as expected, i.e. they eachcapture only their target PCR amplicon. Compared to the B-DNA1 probe theB-DNA2 probe is rather inefficient in capturing its cognate amplicon.The capture efficiency of the B-DNA2 probe, however, can be dramaticallyimproved by substituting 12 of its 13 DNA nucleosides by thecorresponding LNA nucleosides. As shown in FIG. 3 the use of the B-LNA2probe in place of the B-DNA2 probe leads to a more that 10 fold increasein the sensitivity of the assay. At the same time the B-LNA2 retains theability of the unmodified B-DNA2 to efficiently discriminate between therelated and non-related amplicon, underscoring the excellent specificityof LNA-oligos. We conclude that 1) biotin covalently attached to an LNAmodified oligo retains its ability to bind to streptavidin, 2) that LNAmodified oligos works efficiently in a MTP based amplicon capture assayand that 3) LNA offers a means to dramatically improve the performanceof standard DNA oligos in the affinity capture of PCR amplicons.

Example 144

[0373] An LNA substituted oligo is able to capture its cognate PCRamplicon by strand invasion. Two identical sets of 10 l reactions ofamplicon1 or 2 (prepared as in Example 143) were mixed with either 1, 5or 25 pmol of the B-LNA2 capture probe (biotin-GGTGGTTTGTTTG-3′, LNAnucleosides in bold; probe specific for amplicon 2) in 1×SSC (0.15 MNaCl, 15 mM citrate, pH 7.0) in a total volume of 450 μl. One set ofreactions were heated to 95° C. for 5 min in order to denature ampliconsand allowed to cool to 25° C. to facilitate hybridisation between theprobe and the target amplicon strand. The other set of reactions wereleft without denaturation. From each of the reactions 190 μl weretransferred to a streptavidin-coated micro plate (Pierce, cat. no.15124)and incubated for one hour at 37° C. After washing the plate withphosphate buffered saline (PBST, 0.15 M Na⁺, pH 7.2, 0.05% Tween 20,3×300 μl/), 200 μl of peroxidase labelled anti-DIG antibodies were added(Boehringer Manheim, diluted 1:1000 in PBST). Plates were incubated for30 min at 37° C. and washed (PBST, 3×300 μl). Wells were assayed forperoxidase activity by adding 100 μl of substrate solution (0.1 Mcitrate-phosphate buffer pH 5.0, 0.66 mg/ml ortho-pheylenediaminedihydrochloride, 0.012% H₂O₂). The reaction was stopped after 10 min byadding 100 μl H₂SO₄ (0.5 M) and the absorbance at 492 nm was read in amicro plate reader. When amplicons are denaturated prior tohybridisation with the capture probe (FIG. 4A) we observe an efficientand sequence specific amplicon capture similar to that shown in Example143. Increasing the concentration of the B-LNA2 from 1 to 5 pmol leadsto an increase in capture efficiency. A further increase to 25 pmol ofprobe results in a decreased signal. This observation is consistent withsaturation of the available biotin binding sites on the streptavidinMTP. When amplicons are not denaturated prior to hybridisation with thecapture probe (FIG. 4B) we also observe an efficient and sequencespecific amplicon capture. In fact, the data shows that amplicon capturewithout denaturation are as effective and specific as amplicon capturewith denaturation. This strongly indicates that the Bio-LNA2 probe iscapable of binding to its target sequence by strand invasion. To ourknowledge, this constitutes the first example ever of sequence specifictargeting of dsDNA under physiological salt conditions by a mixedpurine/pyrimidine probe. Aside from its potential to significantlysimplify a range of basic research and DNA diagnostic procedures thisunexpected property of LNA modified oligos can be foreseen to be ofmajor importance in the development of efficient new drugs by theantisense, and in particular anti-gene approach.

Example 145

[0374] An LNA substituted oligo, immobilised on a solid surface functionefficiently in the sequence specific capture of a PCR amplicon. Wells ofa streptavidin coated microtiter plate (Boehringer Mannheim) wereincubated for 1 hour with either 5 pmol of the B-DNA2 probe(biotin-GGTGGTTTGTTTG-3′; DNA probe specific for amplicon 2) or theB-LNA2 probe (biotin-GGTGGTTTGTTTG-3′, LNA nucleosides in bold; LNAprobe specific for amplicon 2) in a total volume of 100 μl 1×SSC (0.15 MNaCl, 15 mM citrate, pH 7.0). In total, four wells were incubated withthe B-DNA2 probe, four wells with the B-LNA2 probe and four wells wereincubated with buffer alone. After incubation the wells were washedthree times with 1×SSC. DIG-labelled amplicon1 (60 μl) or amplicon2 (60μl) (prepared as in Example 143) were mixed with 540 μl of 1×SSC, heatdenaturated at 95° C. for 5 min., and transferred (100 μl) to the microplate wells. Two of the wells containing either B-DNA2, B-LNA2 or nocapture probe received amplicon1 and two of the wells containing B-DNA2,B-LNA2 or no capture probe received amplicon2. After 1 hour at 37° C.the plate was washed 3 times with phosphate buffered saline (PBST, 0.15M Na⁺, pH 7.2, 0.05% Tween 20, 3×300 μl) and 200 μl of peroxidaselabelled anti-DIG antibodies were added (Boehringer Mannheim, diluted1:1000 in PBST). Plates were incubated for 30 min at 37° C. and washed 3times with 300 μl PBST. Wells were assayed for peroxidase activity byadding 100 μl of substrate solution (0.1 M citrate-phosphate buffer pH5.0, 0.66 mg/ml ortho-pheylenediamine dihydrochloride, 0.012% H₂O₂). Thereaction was stopped after 6 min by adding 100 μl H₂SO₄ (0.5 M) and theabsorbance at 492 nm was read in a micro plate reader. As shown in FIG.5, the LNA modified capture probe (B-LNA2) captures its specificamplicon (amplicon2) very efficiently and significantly better (approx.five fold increase in sensitivity) than the corresponding unmodified DNAcapture probe (B-DNA2). No signal is obtained when the B-LNA2 probe isincubated with the unrelated amplicon (amplicon1) underscoring theexquisite specificity of the B-LNA2 probe. We conclude that LNA modifiedoligos function efficiently in the sequence specific capture of PCRamplicons when immobilised on a solid surface. We further conclude thatthe use of LNA modified oligos in place of standard DNA oligos providefor a better signal to noise ratio. Thus, LNA offers a means tosignificantly improve the performance of current DNA based assays thatutilises immobilised capture probes, like for instance the array formatwherein multiple immobilised probes are used to simultaneously detectthe occurrence of several different target sequences in a sample.

Example 146

[0375] Fully mixed LNA monomers can be used to significantly increasethe performance of immobilised biotinylated-DNA oligos in the sequencespecific capture of PCR amplicons in a MTP format. Three DIG labelledamplicons from Nras sequence (ref.: Nucleic Acid Research, 1985, Vol.13, No. 14, p 52-55) were generated by PCR amplification as follows:

[0376] PCR primers:

[0377] Forward primer: 5′-CCAGCTCTCAGTAGTTTAGTACA-3′ bases 701-723according to the NAR reference.

[0378] 910 bp reverse primer: 5′-GTAGAGCTTTCTGGTATGACACA-3′ bases1612-1590 (reverse sequence according to NAR ref.).

[0379] 600 bp reverse primer: 5′-TAAGTCACAGACGTATCTCAGAC-3′ bases1331-1308 (reverse sequence according to NAR ref.).

[0380] 200 bp reverse primer: 5′-CTCTGTTTCAGACATGAACTGCT-3′ bases909-886 (reverse sequence according to NAR ref.).

[0381] PCR reaction mixture for Nras amplicons: 2.3 μl human placentalgenomic DNA (440 ng/μl), 50 μl 10×PCR buffer (without MgCl₂ PerkinElmer), 30 μl 25 mM MgCl₂, 50 μl dNTP-mix (2 mM dATP, dCTP, dGTP and 1.8mM dTTP), 10 μl mM Dig-11-dUTP, 10 μl 25 μM forward primer, 10 μl 25 μMreverse primer, 5 μl 5 U/μl AmpliTaq Gold (Perkin Elmer) and water ad500 μl. PCR reaction: The above mixture was made for all the Nras PCRproducts. The only difference being reverse primer 910 bp, 600 bp or 200bp added once at a time. The PCR mixtures were aliquoted into ten PCRtubes each and cycled in a Perkin Elmer 9600 at the followingconditions: 95° C. 3 min; 55° C. 2 min, 72° C. 3 min, 95° C. 1 min (30cycles); 55° C. 2 min, 72° C. 10 min and 4° C. soak. 10 μl from each PCRreaction was analysed on a standard agarose gel and the expectedfragments of approximately 910 bp, 600 bp and 200 bp were observed.Assay conditions: Wells of a streptavidin coated micro-titer plate(Boehringer Mannheim; binding capacity of 20 pmol biotin per well) wereincubated for 1 hour in 5×SSCT (0.75 M NaCl, 75 mM citrate, pH 7.0, 0.1%Tween 20) at 37° C. with either 1 pmol of DNA Nras Cap A(biotin-5′-TTCCACAGCACAA-3′), LNA/DNA Nras Cap A(biotin-5′-TTCCACAGCACAA-3′), LNA Nras Cap A(biotin-5′-TTCCACAGCACAA-3′), DNA Nras Cap B(biotin-5′-AGAGCCGATAACA-3′), LNA/DNA Nras Cap B(biotin-5′-AGAGCCGATAACA-3′) or LNA Nras Cap B(biotin-5′-AGAGCCGATAACA-3′); LNA nucleosides in bold. The Nras Cap Acapture probes capture amplicons Nras 910, Nras 600 and Nras 200. NrasCap B capture probes capture specific amplicons Nras 910 and Nras 600.After incubation with the different capture probes, the wells werewashed in 5×SSCT and 5 μl native or denatured (95° C. 5 min and 10 minon ice) DIG-labelled amplicons (Nras 910, Nras 600 or Nras 200) in 95 μl1×SSCT (0.15 M NaCl, 15 mM citrate, pH 7.0, 0.1% Tween 20) were addedper well and incubated for 1 hour at 37° C. The wells were washed threetimes in phosphate buffered saline (1×PBST, 0.15 M Na⁺, pH 7.2, 0.05%Tween 20) and incubated 30 min at 37° C. with 200 μl peroxidase labelledanti-DIG antibodies (Boehringer Mannheim, diluted 1:1000 in 1×PBST).Finally the wells were washed three times in 1×PBST and assayed forperoxidase activity by adding 100 μl of substrate solution (0.1 Mcitrate-phosphate buffer pH 5.0, 0.66 mg/ml ortho-pheylenediaminedihydrochloride, 0.012% H₂O₂) the reaction was stopped after 9 min byadding 100 μl 0.5 M H₂SO₄ and diluted 4 times in H₂SO₄ before theabsorbance at 492 nm was read in a micro-titer plate reader. As shown inFIG. 23A, capture probes spiked with 12 LNA nucleosides (LNA Nras Cap Aand LNA Cap B) capture very efficiently the specific amplicons withoutprior denaturation (native amplicons). Capture probes spiked with 4 LNAnucleosides (LNA/DNA Nras Cap A and LNA/DNA Nras Cap B) capture the sameamplicons with a lower efficiency and the DNA capture probes (DNA NrasCap A and DNA Nras Cap B) do not capture the specific amplicons at all.The control amplicon, Nras 200, are not captured by the LNA Cap B or theLNA/DNA Nras Cap B probes demonstrating the exquisite specificity of theLNA spiked capture probes. FIG. 23B shows the same experiment performedwith denatured amplicons. Essentially the same picture emerges with theessential difference that capture efficiencies are generally increased.We conclude that LNA modified oligos containing mixed LNA nucleosides(A, T, G or C LNA nucleosides) function efficiently in sequence specificcapture of PCR amplicons when immobilised on a solid surface. We furtherconclude that LNA offers a means to construct capture probes that willfunction efficiently in amplicon capture without prior denaturation i.e.capture by strand displacement. This ability facilitates a significantsimplification of current amplicon detection formats based on DNA.

Example 147

[0382] LNA modified oligos function as primers for nucleic acidpolymerases. The ability of an LNA modified oligo (5′-GGTGGTTTGTTTG-3′,LNA nucleosides in bold) to serve as primer in template dependent,enzymatic elongation were investigated with 3 different classes ofpolymerases. A reverse transcriptase M-MuLV (Boehringer Mannheim) whichcan use both RNA and DNA as template, the Klenow polymerase which isrepresentative of standard DNA polymerases and a thermostablepolymerase, BM-TAQ (Boehringer Mannheim). As control the extensionreactions were conducted using the identical unmodified DNA primer(5′-GGTGGTTTGTTTG-3′). The LNA and DNA primers were labelled with³²P-γ-ATP as previously described in Example 137. A 50 mer DNA oligo(5′-AAAAATCGACGCTCAAGTCAGAAAAGCATCTCACAAACAAACAAACCACC-3′) was used astemplate. The reaction with M-MuL V (Boehringer Mannheim,) contained 2μl of either labelled LNA-primer or DNA primer (10 μM), 2 μl of DNAtemplate (10 μM), 2 μl of 2 mM dNTP, 2 μl of 10×buffer (500 mM Tris-HCl,300 mM KCl, 60 mM MgCl₂, 100 mM DTT, pH 8.3 (37° C.)), 1 μl of enzyme(20U/μl) and water to 20 μl. The reactions were incubated at 37° C. for60 min. The reaction with Klenow polymerase (USB) contained 2 μl ofeither labelled LNA or DNA primer (10 μM), 2 μl of DNA template (10 μM),2 μl of 2 mM dNTP, 2 μl of 10×buffer (100 mM Tris-HCl, 50 mM MgCl₂, 75mM DTT, pH 7.5), 1 μl of enzyme (10U/μl) and water to 20 μl. Thereactions were incubated at 37° C. for 60 min. The reaction with BM-Taq(Boehringer Mannheim) contained 2 μl of either labelled LNA orDNA-primer (10 μM), 2 μl of DNA template (10 μM), 2 μl of 2 mM dNTP, 2μl of 10×buffer (100 mM Tris-HCl, 15 mM MgCl₂, 50 mM KCL, pH 8.3), 1 μlof enzyme (5U/μl) and water to 20 μl. The reactions were incubated at astarting temperature of 37° C. and ramped at 1° C./min to 60° C. wherethey were maintained for 30 min. At the end of the incubation period thereactions were stopped by the addition of 10 μl of loading buffer (0.25%(w/v) bromophenol blue, 0.25% (w/v) xylene cyanol, 80% (v/v) formamid).The samples were heated to 95° C. for 1 min., placed on ice and 2 μl wasloaded onto a 8% sequencing polyacrylamide gel and electrophoresed on aLife Technologies Inc. BRL model 52. After electrophoresis the gel wasdried on the glass plate and subjected to autoradiography (X-ray film:Kodak X-Omat AR). As shown in FIG. 7, clear and similar extensionproducts are observed with both the LNA and DNA primer when either theKlenow polymerase (lanes 3) or the BM-Taq polymerase (lanes 5) is used.

[0383] When M-MuLV reverse transcriptase is used (lanes 2) an extensionproduct can be detected only in the case of the LNA-primer. The labelledLNA and DNA primer that have not been subjected to enzymatic elongationare present in lanes 1, 4 and 6. We conclude that the incorporation ofLNA nucleosides into standard DNA oligos does not prevent recognition ofthe oligo/template duplex by nucleic acid polymerases. We furtherconclude that LNA modified oligos act as efficiently as primers asunmodified DNA oligos.

Example 148

[0384] LNA modified oligo functions as primers in target amplificationprocesses. The ability of LNA modified oligos to act as primers in PCRamplification was analysed with three oligos differing only in thenumber of LNA nucleosides they contained: 4 LNA nucleosides (AL2 primer:5′-GGTGGTTTGTTTG-3′, LNA nucleosides in bold), 1 LNA nucleoside (AL10primer: 5′-GGTGGTTTGTTTG-3′, LNA nucleoside in bold) and no LNAnucleoside (FP2 primer: 5′-GGTGGTTTGTTTG-3′). The PCR reactions (100μl)contained either no template (control), 0.01 ng, 0.1 ng or 1 ng oftemplate (pUC19 plasmid), 0.2 μM reverse primer(5′-GTGGTTCGCTCCAAGCTG-3′), 0.2 μM of either the AL2, AL10 or FP2forward primer, 200 μM of dATP, dGTP, dCTP and dTTP, 10 mM Tris-HCl pH8.3, 1.5 mM MgCl₂, 50 mM KCl and 2.5U of the BM-Taq polymerase. A totalof 50 cycles each consisting of 94° C. 1 min.-45° C. 1 min.-72° C. 1.5min. were conducted (with an additional 2.5U of Taq polymerase addedafter the first 30 cycles) on a Techne Genius thermocycler. After thefinal cycle the reactions were incubated at 72° C. 3 min. and then at 4°C. overnight. To 30 μl of each reaction was added 6 μl of loading buffer(0.25% (w/v) bromophenol blue and 40% (v/v) glycerol) and the samples(together with a Amplisize™ size marker) were loaded onto a 2% agarosegel and electrophoresed for 45 min. at 150V. Finally, the gel wasstained with ethidiumbromid and photographed. As shown in FIG. 8 the PCRreactions using the unmodified forward primer FP2 and unmodified reverseprimer generates detectable amplicons of the correct sizes with allamounts of template used (lane 9: 0.01 ng template, lane 10: 0.1 ng andlane 11: 1 ng). No signal is obtained in the control reaction withouttemplate (lane 12). When the FP2 forward primer is replaced by theprimer containing 1 central LNA nucleoside (AL10) amplicons are alsodetected with all amounts of template used (lane 5: 0.01 ng, lane 6: 0.1ng and lane 7: 1 ng). This clearly indicates that the AL10 primersustains an exponential amplification. i.e. the AL10 primer can be bothextended and used as template in its entirety. Again, the controlreaction without template (lane 8) does not produce an amplicon. Whenthe FP2 forward primer is replaced by the primer containing 4 centralLNA nucleosides (AL2), amplicons of the correct size cannot be detectedin any of the reactions. (lane 1: 0.01 ng template, lane 2: 0.1 ng, lane3: 1 ng and lane 4: no template). With the highest concentration oftemplate (1 ng), however, a high molecular weight band appears in thegel (lane 3). This, however, is an artefact of the RP1 primer asindicated by the control reaction wherein each of the primers AL2 (laneA), AL10 (lane B), FP2 (lane C) and RP1 (lane D) were tested for theirability to produce an amplicon with the highest amount of template (1ng). Since AL2 was shown to act as a primer in Example 147, the absenceof detectable amplicons strongly indicates that it lacks the ability toact as a template, i.e. the block of 4 consecutive LNA nucleosidesblocks the advance of the polymerase thereby turning the reaction into alinear amplification (the product of which would not be detectable bythe experimental set-up used). We conclude that LNA modified oligos canbe used as primers in PCR amplification. We further conclude that thedegree of amplification (graded from fully exponential to linearamplification) can be controlled by the design of the LNA modifiedoligo. We note that the possibility to block the advance of thepolymerase by incorporating LNA nucleosides into the primer facilitatesthe generation of amplicons carrying single stranded ends. Such ends arereadily accessible to hybridisation without denaturation of the ampliconand this feature could be useful in many applications.

Example 149

[0385] An LNA modified oligomer carrying a 5′anthraquinone can becovalently immobilised on a solid support by irradiation and theimmobilised oligomer is efficient in the capture of a complementary DNAoligo. Either 25 pmol/μl or 12.5 pmol/μl of an anthraquinone DNA oligo(5′-AQ-CAG CAG TCG ACA GAG-3′) or an anthraquinone LNA modified DNAoligo (5′-AQ-CAG CAG TCG ACA GAG-3′; LNA monomer is underlined) wasspotted (1 μl/spot) in 0.2 M LiCl on a polycarbonate slide (Nunc). Theoligos were irradiated for 15 min with soft UV light. After irradiationthe slide was washed three times in Milli-Q water and air-dried. 25 mlof 0.5 pmol/μl of complimentary biotinylated oligomer (5′-biotin-CTC TGTCGA CTG CTG-3′) was hybridised to the immobilised oligomers in 5×SSCT(75 mM Citrate, 0.75 M NaCl, pH 7.0, 0.1% Tween 20) at 50° C. for 2hours. After washing four times with 1×SSCT and one time phosphatebuffered saline (PBST, 0.15 M Na⁺, pH 7.2, 0.05% Tween 20), 25 ml PBSTcontaining 0.06 μg/ml streptavidin conjugated horse radish peroxidaseand 1 μg/ml streptavidin were added to the slide. The slide wasincubated for 30 min and washed 4 times with 25 ml PBST. The slide wasvisualised by using chemoluminescent substrate (SuperSignal; Pierce) asdescribed by the manufacturer and X-ray film (CL-XPosure film, Pierce34075). As shown in FIG. 9 both the AQ-DNA oligo and the AQ-LNA modifiedDNA oligo yields a clearly detectable signal. We conclude thatanthraquinone linked LNA modified DNA oligos can be efficiently attachedto a solid surface by irradiation and that oligos attached in this waysare able to hybridise to their complementary target DNA oligos.

Example 150

[0386] Hybridisation and detection on an array with different LNAmodified Cy3-labelled 8 mers. Slide preparation: Glass slides wereaminosilanised using a 10% solution of amino propyl triethoxy silane inacetone followed by washing in acetone. The following oligonucleotideswere spotted out onto the slides: Pens Oligo used Oligo sequence 1 + 2 +3 Sequence cf. probes Seq. 3 5′-GTA TGG AG-3′ 1 pmol/μl 1 internalmismatch Seq. 6 5′-GTA TGA AG-3′ 1 pmol/μl match

[0387] Ten repeat spots, approximately 1 nl each spot, were performedfor each oligonucleotide from each pen on each of 12 slides. Probes (LNAmonomers in bold):

[0388] a) Seq. No.aZ1 5′-Cy3-CTT CAT AC-3′

[0389] b) Seq. No.aZ2 5′-Cy3-CTT CAT AC-3′

[0390] c) Seq. No.aZ3 5′-Cy3-CTT CAT AC-3′

[0391] d) Seq. No.16 5′-Cy3-CTT CAT AC-3′

[0392] Slides and Conditions for Hybridisation:

[0393] Slides 1, 2 and 3 hybridised with aZ1 probe @ 300 fmol/μl, 30fmol/μl, 3 fmol/μl

[0394] Slides 4, 5 and 6 hybridised with aZ2 probe @ 300 fmol/μl, 30fmol/μl, 3 fmol/μl

[0395] Slides 7, 8 and 9 hybridised with aZ3 probe @ 300 fmol/μl, 30fmol/μl, 3 fmol/μl

[0396] Slides 10, 11 and 12 hybridised with seq. 16 probe @ 300 fmol/μl,30 fmol/μl, 3 fmol/μl

[0397] A probe diluted in 30 μl hybridisation buffer (5×SSC, 7% sodiumlauryl sarcosine) was pipetted along the length of each slide, coveredwith a coverslip, and placed into a plastic box on top of a plasticinsert, which was lying on a paper towel wetted with water. Boxes werecovered with aluminium foil to keep light out, and incubated at +4° C.overnight.

[0398] Slide Washes: Coverslips were removed and the slides inserted inracks (6 slides per rack) which were placed in glass slide dishes,wrapped in foil: Slide Number Wash buffer (4° C.) Wash time Probesequence  1, 2, 3 5 × SSC, 0.1% Tween-20 2 × 5 min Seq. No. aZ1  4, 5, 65 × SSC, 0.1% Tween-20 2 × 5 min Seq. No. aZ2  7, 8, 9 5 × SSC, 0.1%Tween-20 2 × 5 min Seq. No. aZ3 10, 11, 12 5 × SSC, 0.1% Tween-20 2 × 5min Seq. No. 16

[0399] After washing, slides were blow-dried and scanned. Thefluorescence was imaged on a slide scanner and the data analysed fromImageQuant software (Molecular Dynamics). As shown in FIG. 11, nobinding of the Cy3 labelled probes is observed to the mismatched oligo 3with either the unmodified probe (slide 10-12), single LNA modifiedprobe aZ1 (slide 1-3) single LNA modified probe aZ2 (slide 4-6) ortriple LNA modified probe aZ3 (slide 7-9) (i.e. the obtained signal withthe mismatched oligo 3 is comparable to the background signal). Withcomplementary oligo 6, specific signals are observed in all cases. Theintensity of these signals clearly correlates with the number of LNAspresent in the probes and with the concentration of the probes. Each LNAT residue approximately increased the signal strength by about a factorof 2 over that of the normal DNA oligo probe, i.e. aZ1 and aZ2=2×signalof sequence 16, and aZ3=8×signal of sequence 16. The match/mismatchdiscrimination is good with the LNA T base replacements, and with theincreased signal strength, the mismatch discriminations appear to beeasier.

Example 151

[0400] Hybridisation and detection of end mismatches on an array withLNA modified Cy3-labelled 8 mers. Slide preparation: Glass slides wereaminosilanised using a 10% solution of amino propyl triethoxy silane inacetone followed by washing in acetone. The following oligonucleotideswere spotted out at 1 pmol/μl onto the slides:

[0401] Seq No.9 5′-GTGTGGAG-3′

[0402] Seq No.15 5′-GTGTGGAA-3′

[0403] Seq No.131 5′-GTGTGGAT-3′

[0404] Seq No.132 5′-GTGTGGAC-3′

[0405] Seq No.133 5′-ATGTGGAA-3′

[0406] Seq No.134 5′-CTGTGGAA-3′

[0407] Seq No.135 5′-TTGTGGAA-3′

[0408] Ten repeat spots, approximately 1 nl each spot, were performedfor each oligonucleotide from each of 6 pens on each of 12 slides.

[0409] Probes (LNA Monomers in Bold):

[0410] DNA

[0411] Probe No.1: 5′-Cy3-TTCCACAC-3′

[0412] Probe No.2: 5′-Cy3-GTCCACAC-3′

[0413] Probe No.3: 5′-Cy3-ATCCACAC-3′

[0414] Probe No.4: 5′-Cy3-CTCCACAC-3′

[0415] Probe No.5: 5′-Cy3-TTCCACAT-3′

[0416] Probe No.6: 5′-Cy3-TTCCACAG-3′

[0417] LNA

[0418] Probe No.35Z-1: 5′-Cy3-TTCCACAC-3′

[0419] Probe No.35Z-2: 5′-Cy3-GTCCACAC-3′

[0420] Probe No.35Z-3: 5′-Cy3-ATCCACAC-3′

[0421] Probe No.35Z-4: 5′-Cy3-CTCCACAC-3′

[0422] Probe No.35Z-5: 5′-Cy3-TTCCACAT-3′

[0423] Probe No.35Z-6: 5′-Cy3-TTCCACAG-3′

[0424] Probes with LNA monomers are prefixed with 35Z- as part of thesequence number. Specific LNA monomers are indicated in italics/bold andare situated at the 3′ and 5′ ends of the LNA oligos.

[0425] Slides and conditions for hybridisation: Each probe sequence washybridised on a separate slide, and all probe concentrations were 1fmol/μl. Each probe was diluted in hybridisation buffer (5×SSC, 7%sodium lauryl sarcosine), of which 30 μl was pipetted along the lengthof each slide, covered with a coverslip, and placed into a plastic boxon top of a plastic insert, which was lying on a paper towel wetted withwater. Boxes were covered with aluminium foil to keep light out, andincubated at +4° C. overnight.

[0426] Slide Washes: Coverslips were removed and the slides inserted inracks (8 slides per rack) which were placed in glass slide dishes,wrapped in foil. All slides were washed in 5×SSC for 2×5 min at +4° C.After washing, slides were blow-dried and scanned. The fluorescence wasimaged on a slide scanner and the data analyzed from ImageQuant software(Molecular Dynamics).

[0427] Conclusions: As shown in FIGS. 12 and 13, probes containing LNAnucleosides at their 5′ and 3′ ends are in the majority of casessignificantly better in discriminating between matched and mismatchedtarget sequences than their corresponding unmodified oligonucleotides.

[0428] For DNA oligos, C=T mismatches were the most difficult todistinguish, for example, where probe sequence 1 hybridised to targetsequence 132 and where probe sequence 5 hybridised to target sequence134. Other mismatches were visible such as T=T and G=T mismatches, butthese spots were less intense, for example where probe sequences 5 and 6respectively hybridised to target sequence 135. The LNA oligos,significantly reduced these C=T and T=T mismatch spot intensites, tocomparable levels to other mismatches. The relative spot intensities ofprobe sequences 1, 2 and 3 were similar for the DNA and LNA oligos.However, with probe sequences 4, 5 and 6, the LNA oligos gave asignificantly increased spot intensity when hybridised to their matchtarget sequences 9, 133 and 134 respectively.

Example 152

[0429] Hybridization and detection of end mismatches on an array with ATand all LNA modified Cy3-labelled 8 mers. Slide preparation: Glassslides were aminosilanized using a 10% solution of amino propyltriethoxy silane in acetone followed by washing in acetone. Thefollowing oligonucleotides were spotted out at 1 pmol/μl onto theslides:

[0430] Seq No.9 5′-GTGTGGAG-3′

[0431] Seq No.15 5′-GTGTGGAA-3′

[0432] Seq No.131 5′-GTGTGGAT-3′

[0433] Seq No.132 5′-GTGTGGAC-3′

[0434] Seq No.133 5′-ATGTGGAA-3′

[0435] Seq No.134 5′-CTGTGGAA-3′

[0436] Seq No.135 5′-TTGTGGAA-3′

[0437] Ten repeat spots, approximately 1 nI each spot, were performedfor each oligonucleotide from each of 6 pens on each of 36 slides.

[0438] Probes: (LNA Monomers in Bold):

[0439] DNA:

[0440] Probe No.1: 5′-Cy3-TTCCACAC-3′

[0441] Probe No.2: 5′-Cy3-GTCCACAC-3′

[0442] Probe No.3: 5′-Cy3-ATCCACAC-3′

[0443] Probe No.4: 5′-Cy3-CTCCACAC-3′

[0444] Probe No.5: 5′-Cy3-TTCCACAT-3′

[0445] Probe No.6: 5′-Cy3-TTCCACAG-3′

[0446] AT LNA:

[0447] Probe No.ATZ-1: 5′-Cy3-TTCCACAC-3′

[0448] Probe No.ATZ-2: 5′-Cy3-GTCCACAC-3′

[0449] Probe No.ATZ-3: 5′-Cy3-ATCCACAC-3′

[0450] Probe No.ATZ-4: 5′-Cy3-CTCCACAC-3′

[0451] Probe No.ATZ-5: 5′-Cy3-TTCCACAT-3′

[0452] Probe No.ATZ-6: 5′-Cy3-TTCCACAG-3′

[0453] All LNA:

[0454] Probe No.AIIZ-1: 5′-Cy3-TTCCACAC-3′

[0455] Probe No.AIIZ-2: 5′-Cy3-GTCCACAC-3′

[0456] Probe No.AIIZ-3: 5′-Cy3-ATCCACAC-3′

[0457] Probe No.AIIZ-4: 5′-Cy3-CTCCACAC-3′

[0458] Probe No.AIIZ-5: 5′-Cy3-TTCCACAT-3′

[0459] Probe No.AIIZ-6: 5′-Cy3-TTCCACAG-3′

[0460] Probes with LNA monomers are prefixed with ATZ- or AIIZ- as partof the sequence number. Specific LNA monomers are indicated in italicsfor the LNA oligos.

[0461] Slides and conditions for hybridization: Each probe sequence washybridized on a separate slide, and all probe concentrations were 1fmol/μl. Each probe was diluted in hybridization buffer (5×SSC, 7%sodium lauryl sarcosine), of which 30 μl was pipetted along the lengthof each slide, covered with a coverslip, and placed into a plastic boxon top of a plastic insert, which was lying on a paper towel wetted withwater. Boxes were covered with aluminium foil to keep light out, andincubated at room temperature overnight.

[0462] Slide Washes: Coverslips were removed and the slides inserted inracks (9 slides per rack) which were placed in glass slide dishes,wrapped in foil. All slides were washed in 5×SSC for 2×5 minutes at RT.After washing, slides were blow-dried and scanned. The fluorescence wasimaged on a slide scanner and the data analyzed from ImageQuant software(Molecular Dynamics).

[0463] Conclusion: As shown in FIGS. 15A, 15B and 15C, The averageintensity of DNA hybridization at room temperature was about 10% of theintensity achieved with the AT or all LNA modified oligos. No spots wereseen on slides hybridized with DNA probes 5 and 6. These conditions weretherefore not optimal for the DNA probes. However, the match/mismatchdiscrimination is very good with the LNA nucleoside replacements at theA and T bases. The stringency for the all LNA oligos may not be greatenough as the match/mismatch discrimination was not as good as for theAT LNA oligos.

[0464] The oligos with LNA modifications worked very well, and themismatches that were the most difficult to discriminate were;

[0465] Probe 1 to target 135=CT mismatch

[0466] Probe 2 to target 131=GT mismatch

[0467] Probe 3 to target 15=AA mismatch

[0468] Probe 4 to target 131=CT mismatch

[0469] Probe 5 to target 135 TT mismatch

[0470] Probe 6 to target 135 GT mismatch

[0471] Probe 6 to target 133 GA mismatch

[0472] The AT LNA oligos gave good discrimination where these mismatchspot intensities were typically at the most 50% of the intensity of thematch spots. For these mismatches, the all LNA oligos gave mismatch spotintensities about 50 to 70% of the match spot intensities. Overall, LNAmodifications allows the use of higher temperatures for hybridizationsand washes, and end mismatches can be discriminated. These results areat least as good as those from DNA probes hybridised at 4° C. (seeexample 151).

Example 153

[0473] Use of [α³³P] ddNTP's and ThermoSequenase™ DNA Polymerase toSequence DNA Templates Containing LNA T Monomers. Radiolabelledterminator sequencing reactions were set up in order to test the abilityof the LNA T monomer to be accepted as a template-for DNA polymerases.The 15 mer primer (sequence: 5′-TGC ATG TGC TGG AGA-3′) was used toprime the following short oligonucleotide sequences (LNA monomer inbold):

[0474] Template 1 3′-ACG TAC ACG ACC TCT ACC TTG CTA-5′

[0475] TemplateTZ1 3′-ACG TAC ACG ACC TCT ACC TTG CTA-5′

[0476] The following reaction mixes were made:

[0477] Template 1 mix:

[0478] 2 μl ×16 ThermoSequenase Buffer

[0479] 6 μl Primer 2 pmole/μl

[0480] 6 μl Template 11 pmole/μl

[0481] 4 μl Water

[0482] 2 μl ThermoSequenase DNA Polymerase (4U/μl)

[0483] 20 μl Total volume

[0484] Template TZ1 mix

[0485] 2 μl ×16 ThermoSequenase Buffer

[0486] 6 μl Primer 2 pmole/μl

[0487] 6 μl Template TZ11 pmole/μl

[0488] 4 μl Water

[0489] 2 μl ThermoSequenase DNA Polymerase (4U/μl)

[0490] 20 μl Total volume

[0491] 2 μl Nucleotide mix (7.5 μM each dNTP) was added to each of 8Eppendorf tubes. 0.5111 [α³³P] ddATP was added to tubes 1 and 5. 0.5 μl[α³³P] ddCTP was added to tubes 2 and 6. 0.5 μl [α³³P] ddGTP was addedto Tubes 3 and 7. 0.5 μl [α³³P] ddTTP was added to tubes 4 and 8. 4.5 μlof Template 1 mix was added to each of tubes 1-4. 4.5 μl of Template TZ1mix was added to each of tubes 5-8. All the reactions were incubated at60° C. for 3 min. The reactions were stopped by the addition of 41formamide/EDTA stop solution. Reactions were heated at 95° C. for 3 minbefore loading onto a 19% polyacrylamide 7M urea gel. The gel was fixedin 10% acetic acid 10% methanol before transferring to 3 MM paper anddrying. The dried gel was exposed to Kodak Biomax autoradiography film.

[0492] The results are depicted in FIG. 18 (track 1-4) and FIG. 19(5-8). The tracks correspond to the following reactions: (FIG. 18): Lane1—ddATP track. Lane 2—ddCTP track. Lane 3—ddGTP track, Lane 4—ddTTPtrack. Lane 5—8-32 base oligo markers; FIG. 19: Lane A—8-32 base oligomarkers. Lane 5—ddATP track. Lane 6—ddCTP track. Lane 7—ddGTP track.Lane 8—ddTTP track.

[0493] As is evident from FIGS. 18 and 19, the full sequence of bothtemplates can easily be read from the autorad. The sequence is 5′-TGGAAC GTA-3′ which corresponds to the template sequence 3′-ACC TTG CTA-5′.This shows that a single LNA T monomer can act as a template for DNApolymerases. The LNA T monomer is specifically copied as “T” with ddATPbeing incorporated.

[0494] Therapeutic Applications

Example 154

[0495] LNA modified oligos can be transferred into cells. Experimentwith radiolabelled LNA oligos. 10 pmol of a oligodeoxynucleotide (ODN)(ODN#10: 5′-TTA ACG TAG GTG CTG GAC TTG TCG CTG TTG TAC TT-3′, a 35-mercomplementary to human Cathepsin D) and 10 pmoles of two LNA oligos:AL16 (5′-d(TGT GTG AAA TTG TTA T)-3′, LNA nucleosides in bold) and AL17(5′-d(ATA AAG TGT AAA G)-3′, LNA nucleosides in bold) were mixed with T4polynucleotide Kinase (10 units, BRL cat. no. 510-8004SA), 5 μlgamma-32P-ATP 5000 Ci/mmol, 10 uCi/μl (Amersham) in kinase buffer (50 mMTris/HCl pH 7, 6, 10 mM MgCl₂, 5 mM DTT, 0.1 mM EDTA). The samples wereincubated for 45 min at 37° C. and afterwards heated to 68° C. for 10min, and then moved to +0° C. Unincorporated nucleotides were removed bypassage over Chroma Spin TE-10 columns (Clontech cat. no. K1320-1). Theyields were 5×10⁵ cpm/μl, 2×10⁵ cpm/μl and 0.8×10⁵ cpm/μl for ODN#10,AL16 and AL17, respectively. MCF-7 human breast cancer cells originallyobtained from the Human Cell Culture Bank (Mason Research Institute,Rockville) were cultured in DME/F12 culture medium (1:1) supplementedwith 1% heat inactivated fetal calf serum (Gibco BRL), 6 ng/ml bovineinsulin (Novo) and 2.5 mM glutamax (Life Technologies) in 25 cm² cellculture flasks (Nunclon, NUNC) and incubated in a humified incubator at37° C., 5%CO₂, 20%02, 75%N₂. The MCF-7 cells were approximately 40%confluent at the time of the experiment. A small amount (less than 0.1pmol) of the kinased oligos were mixed with 1.5 μg pEGFP-NI plasmid(Clontech cat. no. 60851) and mixed with 100 μl diluted FuGENE6transfection agent (Boehringer.Mannheim cat no. 1 814 443), dilution: 5μl FuGENE6 in 95 μl DME/F12 culture medium without serum. TheFuGENE6/DNA/oligo-mixture were added directly to the culture medium (5ml) of adherent growing MCF-7 cells and incubated with the cells for 18hours, closely following the manufacturers directions. Three types ofexperiments were set up. 1) ODN#10+pEGFP-NI; 2) AL16+PEGFP-NI; 3)AL17+pEGFP-NI. Cellular uptake of DNA/LNA material were studied byremoving FuGENE6/DNA/oligo-mixture containing medium (an aliquot wastransferred to a scintillator vial). Cells were rinced once withphosphate buffered saline (PBS), fresh culture medium was added andcells inspected by fluorescence microscopy. Approximately 30% of thetransfected cells contained green fluorescent material, indicating thatapproximately 30% of the cells have taken up the pEGFP-NI plasmid andexpressed the green fluorescent protein coded by this plasmid. Followingfluorescence microscopy the adherent MCF-7 cells were removed from theculture flasks. Briefly, the culture medium was removed, then cells wererinsed with a solution of 0.25% trypsin (Gibco BRL) 1 mM EDTA in PBS(without Mg²⁺ and Ca²⁺), 1 ml trypsin/EDTA was added and cells wereincubated 10 min at 37° C. During the incubation the cells loosened andwere easily resuspended and transferred to scintillator vials. The cellswere then completely dissolved by addition of 10 ml Optifluorscintillation coctail (Packard cat. no. 6013199), and the vials werecounted in a Wallac 1409 scintillation counter. The results were asfollows: 1) ODN#10+pEGFP-NI: approximately 1.4% of the addedradioactivity were associated with cellular material; 2) AL16+pEGFP-NI:approximately 0.8% of the added radioactivity were associated withcellular material; and 3) AL17+pEGFP-NI: approximately 0.4% of the addedradioactivity were associated with cellular material. We conclude that0.4-0.8% of the added LNA oligos were taken up by the cells.

Example 155

[0496] LNA is efficiently delivered to living human MCF-7 breast cancercells. To increase the efficiency of LNA-uptake by human MCF-7 cellsdifferent transfection agents were tested with various concentrations of5′FITC-labelled LNAs and DNA. The oligonucleotides described in thetable below were tested. TABLE Oligonucleotides tested Name Sequence(LNA monomers in bold) Characteristics AL16 5′-TGT GTG AAA TTG TTA T-3′LNA, enzym. FITC- labeled AL17 5′-ATA AAG TGT AAA G-3′ LNA, enzym. FITC-labeled EQ3009-01 5′-TGC CTG CAG GTC GAC T-3′ LNA-FITC-labeled EQ3008-015′-TGC CTG CAG GTC GAC T-3′ DNA-FITC-labeled

[0497] AL16 and AL17 were enzymatically labelled with FITC as describedin Example 128. EQ3009-01 and EQ3008-01 were labelled with FITC bystandard solid phase chemistry. Three transfection agents were tested:FuGENE-6 (Boehringer Mannheim cat. no. 1 814 443), SuperFect (Quiagencat. no. 301305) and Lipofectin (GibcoBRL cat. no. 18292-011). HumanMCF-7 breast cancer cells were cultured as described previously (Example154). Three days before the experiments the cells were seeded at a celldensity of approx. 0.8×10⁴ cells per cm². Depending on the type ofexperiment the MCF-7 cells were seeded in standard T25 flasks (Nunc,LifeTechnologies cat. no. 163371A), 24 wells multidish (Nunc,LifeTechnologies cat. no. 143982A) or slide flasks (Nunc,LifeTechnologies cat. no. 170920A). The experiments were performed whencells were 30-40% confluent. Cellular uptake of LNA and DNA was studiedat serum-free conditions, i.e. the normal serum containing DME/F12medium was removed and replaced with DME/F12 without serum before thetransfection-mixture was added to the cells. Under these conditionsSuperFect proved to be toxic to the MCF-7 cells. Transfection mixturesconsisting of SuperFect and either plasmid DNA (pEGFP-N1, Clontech cat.no. 6085-1), oligo DNA or oligo LNA was equally toxic to MCF-7 cells. Incontrast to SuperFect, FuGene6 and Lipofectin worked well with plasmidDNA (pEGFP-N1). However, only lipofectin was capable of efficientdelivery of oligonucleotides to living MCF-7. Briefly, efficientdelivery of FITC-labelled LNA and DNA to MCF-7 cells was obtained byculturing the cells in DME/F12 with 1% FCS to approx. 40% confluence.The Lipofectin reagent was then diluted 40×in DME/F12 medium withoutserum and combined with the oligo to a concentration of 750 nM oligo.The oligo-Lipofectin complex was allowed to form for 15 min at r.t., andfurther diluted with serum-free medium to at final concentration of 250nM oligo, 0.8 ug/ml Lipofectin. Then, the medium was removed from thecells and replaced with the medium containing oligo-Lipofectin complex.The cells were incubated at 37° C. for 6 hours, rinsed once with DME/F12medium without serum and incubated for a further 18 hours in DME/F12with 1% FCS at 37° C. The result of the experiment was evaluated eitherdirectly on living cells in culture flasks or in 24 wells multidishes oron cells cultured in slide flasks and fixed in 4% ice-cold PFA. In allcases a Leica DMRB fluorescence microscope equipped with a highresolution CCD camera was used. The result with living cells is shown inFIG. 16 and the result with fixed cells cultured in slide flask is shownin FIG. 17. Both the cells in FIGS. 16 and 17 was transfected with theFITC-labelled AL16 LNA molecule. By counting total number of cells andgreen fluorescent cells in several fields we observe that FITC-labelledAL16 LNA was transfected into approximately 35% of the MCF-7 cells.Importantly, we saw that the LNA predominantly was localised in thenuclei of the cells (FIG. 17). This is noteworthy, since nuclear uptakeof fluorescent oligos correlates with their antisense activity (Stein C.A. et al. (1997) Making sense of antisense: A debate. In HMS Beagle: ABioMedNet Publication (http://hmsbeagle.com/06/cutedge/overwiev.htm)).Increasing the amount of oligo and lipofectin up to a finalconcentration of 1250 nM oligo and 4 ug/ml lipofectin only increased thepercentage of green fluorescent cells marginally. Increasing theconcentration even further was toxic for the cells. Similar results wereobtained with the other LNAs and the FITC-labelled oligo DNA (see thetable above). We conclude that: 1) LNA can be efficiently delivered toliving MCF-7 breast cancer cells by Lipofectin-mediated transfection. 2)A consistent high fraction, 30% or more of cells, is transfected using afinal concentration of 250 nM LNA, 0.8 ug Lipofectin pr. ml growthmedium without serum. Increasing the concentrations of LNA andLipofectin up to 5 times only increased the transfection yieldmarginally. 3) The procedure transfected the LNA into the nuclei of thecells, which according to literature is a good indication that suchtransfected LNAs may exhibit antisense effects on cells.

Example 156

[0498] LNA modified oligos can be transferred into cells. Experimentwith fluorescein labelled LNA oligos. Two LNA oligos: AL16 (5′-TGT GTGAAA TTG TTA-3′, LNA nucleosides in bold) and AL17 (5′-ATA AAG TGT AAAG-3′, LNA nucleosides in bold) were labeled with fluorescein asdescribed in Example 128. MCF-7 human breast cancer cells were culturedas described in Example 154. Three types of experiments were set up. 1)approximately 1.5 μg FITC-labelled AL16; 2) approximately 1.5 μgFITC-labelled AL17; and 3) approximately 0.75 μg FITC-labelled AL16 and0.75 μg pRSVβgal plasmid (a plasmid expressing the bacterial lac Z genecoded enzyme β-galactosidase, Tulchinsky et. al. (1992) PNAS, 89,9146-50). The two LNA oligos and the LNA-plasmid mix were mixed withFuGENE6 and added to MCF-7 cells as described in Example 154. Afterincubation for 18 hours cellular uptake of the LNA oligos were assessedby fluorescence microscopy of the cell cultures. A part of the treatedcells contained green fluorescent material (see FIG. 16), indicatingthat cells take up the fluorescein labelled LNA. The fluoresceinlabelled AL16 appeared superior to fluorescein labelled AL17 in thisrespect. After fluorescence microscopy the culture medium were removedfrom the cells treated both with fluorescein labelled AL16 and pRSVβgal.The cells were washed once with PBS, fixed in 2% (v/v) formaldehyde,0.2% (v/v) glutaraldehyde at 4° C. for 5 min and β-galactosidasecontaining cells were stained blue with X-gal (5-bromo-4-chloro-3-indoylβ-D-galactopyranosid) which turns from colorless to blue in the presenceof β-galactosidase activity. The X-gal staining showed that the pRSVβgaleffectively had been transferred into cells. We conclude that thefluorescein LNA oligos were taken up by the cells.

Example 157

[0499] LNA modified oligos are relatively stable under cell cultureconditions. Following fluorescence microscopy as described in Example156 cells treated only with the fluorescein labelled AL16 LNA wereallowed to incubate for an additional 3 days. During this period of timethe number of green fluorescent cells appeared unaltered. We concludethat fluorescein labelled LNA oligos has a good stability under theconditions prevailing in cell culture.

Example 158

[0500] Blockade by Antisense Locked Nucleic Acids (LNA) of[D-Ala2]Deltorphin-Induced Antinociception in the Warm Water Tail FlickTest in Conscious Rats. Male Sprague-Dawley rats (300 g) were implantedwith an intrathecal (i.th). polyethylene catheter and allowed to recoverfor at least 5 days before start of injections (including controls). Theantisense LNA compounds (12.5 and 2.5 μg per injection) wereadministered in a 5 μl volume twice-daily (08.00 and 17.00 h) for 3days. No signs of non-specific effects or toxicity could be detected, asshown by observations of locomotor behavior and measurements of bodyweight. The day after the last injection the rats were injected with[D-Ala2]deltorphin (60 μg, i.th) and tested in the warm water (52° C.)tail flick test for 6 opioid receptor-mediated antinociception. Data arepresented in FIG. 14 as medians based on 6-8 animals per group (dataconverted to percent maximum possible response, % MPE). Statisticalanalyses were performed by means of Kruskal-Wallis 1-way ANOVA by ranks,followed by comparisons of treatments versus control. As shown in FIG.14, deltorphin produced a robust antinociceptive effect insaline-treated controls. This response was statistically significantlysuppressed in both antisense LNA groups (12.5 and 2.5 μg) as comparedwith saline-treated controls.

[0501] LNA Solid Supports

Example 159

[0502] General method for DMT-LNA nucleoside succinates. Base protectedDMT-LNA nucleoside and succinic anhydride (1.5 equivalents) were takenin anhydrous ethylene dichloride (10 ml/g of nucleoside). To themixture, triethylamine (2 equivalents) was added and the mixture wasstirred at room temperature. Reaction was followed by HPLC (conditionssame as for tritylation). After complete reaction (>95%), reactionmixture was concentrated, coevaporated with ethylene dichloride andacetonitrile, and dried in vacuo to remove triethylamine. Residue wasdissolved in ethylene dichloride or ethyl acetate (˜100 ml/g of startingnucleoside), washed with cold 10% citric acid (3×80 ml/g) and cold water(3×80 ml/g). Organic layer was dried over anhydrous sodium sulfate,filtered and concentrated with or without addition of 1-2 equivalents oftriethylamine. Residual solid was coevaporated with anhydrousacetonitrile (2-3×) and dried in vacuo to give pure product as whitesolid.

[0503] General method for LNA nucleoside supports. Base protectedDMT-LNA-nucleoside succinate (free acid or triethylammonium salt, 65micromol/g of support), amino derivatised support (Primer Support™ 30HL,160 micromol amino groups/g of support), DMAP (3 mg/g of support) and1-(3-[dimethylamino]propyl)-3-ethylcarbodimide hydrochloride (80 mg/g ofsupport) were taken in anhydrous pyridine (6 ml/g of support). To thismixture, triethylamine (16 microliter/g of support) was added and themixture was kept on a shaker at 150 rpm overnight. Support was filtered,washed with methanol (3×10 ml/g of support) and dichloromethane (3×10ml/g of support). After air drying, support was dried in vacuo for 0.5h. To this 6% DMAP in anhydrous acetonitrile (Cap A, ˜3 ml/g of support)and a mixture of 20% acetic anhydridel 30% 2,4,6-collidine/50%acetonitrile (Cap B, ˜3 ml/g of support) were added. The micture waskept on shaker for 5 h. Support was filtered, washed with anhydrousdichloromethane (2×10 ml/g of support) and dried as above. It wasresuspended in a mixture of Cap A and Cap B (total vol. 6 ml/g ofsupport) and kept on shaker overnight. Support was filtered, washed withmethanol (6×10 ml/g of support), dichloromethane (3×10 ml/g of support)and dried in air. It was further dried in vacuo for 5-6 h. Loading wasdetermined by dimethoxytrityl assay and was found to be approx. 40μmol/g.

Example 160

[0504] First Strand cDNA Synthesis Using Poly dT Primers Containing LNAT monomers. Reactions were set up in order to test the ability of polydT primers containing LNA T residues to prime 1^(st) strand cDNAsynthesis. The following poly dT primers were tested (LNA monomers arein bold):

[0505] RTZ1 5′-TTTTTTTTTTTTT-3′

[0506] RTZ2 5′-TTT TTT TTT TTT TT-3′

[0507] RTZ3 5′-TTT TTT TTT TTT TT-3′

[0508] RTZ4 5′-TTT TTT TTT TTT TT-3′

[0509] RTZ5 5′-TTT TTT TTT T-3′

[0510] Anchored poly dT primer from RPKO140 kit Cy Dye cDNA labellingkit (Amersham Pharmacia Biotech) was as a control.

[0511] Reactions were set up as follows for each of the primers above:

[0512] 1 μl Arabidopsis mRNA 0.5 μg/μl

[0513] 2 μl poly dT primer 8 pmoles/μl

[0514] 4 μl ×5 AMV Reverse Transcriptase buffer

[0515] 1 μl Water

[0516] 8 μl Total volume

[0517] This mix was then heated to 75° C. for 3 min and then allowed tocool at room temperature for at least 10 min.

[0518] The following was then added to each of the reactions:

[0519] 1 μl 80 mM Sodium Pyrophosphate

[0520] 1 μl Human Placental Ribonuclease Inhibitor 20U/μl

[0521] 7 μl 0.5 mM dNTP solution

[0522] 2 μl [α³³P] dATP 10 mCi/ml 3000 Ci/mmole

[0523] 1 μl AMV Reverse Transcriptase 20U/μl

[0524] 20 μl Total volume

[0525] The reactions were incubated at 42° C. for 2 hours. The reactionswere then heated at 95° C. for 3 min before loading onto a 6%polyacrylamide 7M urea gel. The gel was fixed in 10% acetic acid/10%methanol before transferring to 3 MM paper and drying. The dried gel wasexposed to Kodak Biomax autoradiography film overnight.

[0526] The autoradiograph clearly showed that the LNA containingoligonucleotide primers RTZ1-4 were able to efficiently prime cDNAsynthesis. The amount and intensity of the cDNA products produced inthese reactions was equal to that produced with the anchored poly dTcontrol oligonucleotide. RTZ 5 did produce some cDNA, but the yield wassignificantly lower than that produced with the control oligo primer.

Example 161

[0527] LNA-modified oligonuclotides covalently attached to Separosebeads function efficiently in the sequence specific capture of RNAmolecules. Three oligos were synthesised by chemistry (Amy Mueller) forevaluation in poly (rA) binding. NH₂(T8)-T Control NH₂(T15)-T ControlNH₂(LNA-T8)-T Test

[0528] 200 mmol of each oligo were coupled to 50 mg of preparedCNBr-activated Separose B (Pharmacia) per booklet instructions.Unreacted binding sites on the resin were blocked in 100 nM Tris pH 8.0.Table of Oligo Binding Data T9 oligo T16 oligos LNA T9 oligo No oligoSteps A₂₆₀ units A₂₆₀ units A₂₆₀ units Control Oligo reacted 14.7 26.014.7 0 (200 nM) (200 nM) (200 nM) Unbound oligo 5.50 10.43 4.20 — ∴Boundoligo 9.20 15.57 10.50 — % Bound 62.6% 59.9% 71.4% —

[0529] Oligo bound resins were divided into two portions (25 mg resineach) for, poly (rA) binding analysis in duplicate. Poly (rA) Pharmacia#27-4110-01 (dissolved at 28.2 A₂₆₀ units/ml in binding buffer) was usedfor binding. Five (5) A₂₆₀ units were bound to duplicate 25 mg portionsof each oligo bound resin per SOP QC 5543. Unbound “breakthrough” poly(rA) was quantitated by A₂₆₀ absorbance and used to calculate bound. Thefate of the bound poly (rA) was tracked through Low salt buffer wash andseveral elutions. As shown in Table 10 both the LNA and DNA coated beadsfunction efficiently in the capture of poly (rA) target molecules. TheLNA coated beads, however, bind the poly (rA) target much more tightlythan the DNA coated beads as evidenced by the poly (rA) elution profilesof the different beads. We conclude that 1) an LNA T9 oligo is efficientin the capture of RNA molecules containing a strech of A residues andthat 2) the captured RNA molecules are bound much more tightly to theLNA T9 oligo beads than to the control DNA T9 and DNA T16 oligo. TABLE 1Monomer Z T_(m) T_(m) (° C.) T_(m) (° C.) T_(m) (° C.) Oligo Target No.Na₂HPO₄/EDTA Na₂HPO₄/NaCl/EDTA Na₂HPO₄/TMAC 5′-d(GTGATATGC)-3′5′-d(GCATATCAC)-3′  1 28 42 5′-d(GCATTTCAC)-3′  2 12 315′-d(GCATGTCAC)-3′  3 19 23 5′-d(GCATCTCAC)-3′  4 11 305′-d(GCATAACAC)-3′  5 12 5′-d(GCATAGCAC)-3′  6 <10 5′-d(GCATACCAC)-3′  7<10 5′-(GCAUAUCAC)-3′  8 28 5′-(GCAUCUCAC)-3′  9 10 5′-d(GTGATATGC)-3′5′-d(GCATATCAC)-3′ 10 44 56 5′-d(GCATTTCAC)-3′ 11 27 435′-d(GCATGTCAC)-3′ 12 30 43 5′-d(GCATCTCAC)-3′ 13 23 385′-d(GCATAACAC)-3′ 14 28 5′-d(GCATAGCAC)-3′ 15 28 5′-d(GCATACCAC)-3′  15A 29 5′-(GCAUAUCAC)-3′ 16 50 5′-(GCAUCUCAC)-3′ 17 335′-d(GTGAGATGC)-3′ 5′-d(GCATATCAC)-3′ 18 26 39 5′-d(GCATTTCAC)-3′ 19 3344 5′-d(GCATGTCAC)-3′ 20 28 38 5′-d(GCATCTCAC)-3′ 21 49 575′-d(GCATAACAC)-3′ 22 <15 5′-d(GCATAGCAC)-3′ 23 <15 5′-d(GCATACCAC)-3′24 <15 5′-(GCAUAUCAC)-3′   24A 34 5′-(GCAUCUCAC)-3′   24B 595′-d(GTGAUATGC)-3′ 5′-d(GCATATCAC)-3′ 25 44 56 5′-d(GCATTTCAC)-3′ 26 2544 5′-d(GCATGTCAC)-3′ 27 32 43 5′-d(GCATCTCAC)-3′ 28 24 375′-d(GCATAACAC)-3′ 29 27 5′-d(GCATAGCAC)-3′ 30 28 5′-d(GCATACCAC)-3′ 3120 5′-d(GTGAGATGC)-3′ 5′-d(GCATATCAC)-3′ 32 17 34 5′-d(GCATTTCAC)-3′ 3316 30 5′-d(GCATGTCAC)-3′ 34 15 28 5′-d(GCATCTCAC)-3′ 35 33 445′-d(GCATAACAC)-3′ 36 9.0 5′-d(GCATAGCAC)-3′ 37 <5 5′-d(GCATACCAC)-3′ 38<5 5′-(GCAUCUCAC)-3′   38A 33 5′-d(GGTGGTTTGTTTG)-3′5′-d(CAAACAAACCACA)-3′ 39 31 47 55 5′-(CAAACAAACCACA)-3′   39A 32 525′-d(GGTGGTTTGTTTG)-3′ 5′-d(CAAACAAACCACA)-3′ 40 40 57 675′-(CAAACAAACCACA)-3′   40A 50 70 d(GGTGGTTTGTTTG)-3′5′-d(CAAACAAACCACA)-3′ 41 67 83 >90 5′-(CAAACAAACCACA)-3′   41A 85 >935′-d(TTTTTTTTTTTTTT)-3′ 5′-d(AAAAAAAAAAAAAA)-3′ 42 365′-(AAAAAAAAAAAAAA)-3′ 43 32 5′-d(TTTTTTTTTTTTTT)-3′5′-d(AAAAAAAAAAAAAA)-3′ 44 36 5′-(AAAAAAAAAAAAAA)-3′ 45 325′-d(TTTTTTTTTTTTTT)-3′ 5′-d(AAAAAAAAAAAAAA)-3′ 46 345′-(AAAAAAAAAAAAAA)-3′ 47 40 5′-d(TTTTTTTTTTTTTT)-3′5′-d(AAAAAAAAAAAAAA)-3′ 48 42 5′-(AAAAAAAAAAAAAA)-3′ 49 525′-d(TTTTTTTTTTTTTT)-3′ 5′-d(AAAAAAAAAAAAAA)3′ 50 475′-(AAAAAAAAAAAAAA)-3′ 51 53 5′-d(TTTTTTTTTT)-3′ 5′-d(AAAAAAAAAAAAAA)-3′52 80 5′-(AAAAAAAAAAAAAA)3′ 53 70 5′-d(AAAACAAAA)-3′ 54 635′-d(AAAAGAAAA)-3′ 55 55 5′-d(AAAATAAAA)-3′ 56 65 5′-d(GTGAAATGC)-3′5′-d(GCATATCAC)-3′ 57 26 5′-d(GCATTTCAC)-3′ 58 45 5′-d(GCATGTCAC)-3′ 5923 5′-d(GCATCTCAC)-3′ 60 25 5′-d(GTGA^(Me)CATGC)-3′ 5′-d(GCATATCAC)-3′61 <15 5′-d(GTGA^(Me)CATGC)-3′ 5′-d(GCATATCAC)-3′ 63 325′-d(GCATTTCAC)-3′ 64 27 5′-d(GCATGTCAC)-3′ 65 53 5′-d(GCATCTCAC)-3′ 6632 5′-d(GTGACATGC)-3′ 5′-d(GCATATCAC)-3′ 67 32 5′-d(GCATGTCAC)-3′ 69 525′-d(GTGATATG^(Me)C)-3′ 5′-d(GCATATCAC)-3′ 71 64 5′-d(GCATGTCAC)-3′ 7352 5′-(GCAUAUCAC)-3′ 75 74 5′-(GCAUCUCAC)-3′ 76 60 5′-d(CACTATACG)-3′ 7740 5′-d(GTGTTTTGC)-3′ 5′-d(GCAAAACAC)-3′ 78 52

[0530] TABLE 2 Monomer V T_(m) T_(m) (° C.) T_(m) (° C.) T_(m) (° C.)Oligo Target No. Na₂HPO₄/EDTA Na₂HPO₄/NaCl/EDTA Na₂HPO₄/TMAC5′-d(TTTTTTTTTTTTTT)-3′ 5′-d(AAAAAAAAAAAAAA)-3′ 325′-(AAAAAAAAAAAAAA)-3′ 27 5′-d(TTTTTTTTTTTTTT)-3′5′-d(AAAAAAAAAAAAAA)-3′ 31 5′-(AAAAAAAAAAAAAA)-3′ 285′-d(TTTTTTTTTTTTTT)-3′ 5′-d(AAAAAAAAAAAAAA)-3′ 305′-(AAAAAAAAAAAAAA)-3′ 23 5′-d(TTTTTTTTTTTTTT)-3′5′-d(AAAAAAAAAAAAAA)-3′ 23 5′-(AAAAAAAAAAAAAA)-3′ 315′-d(TTTTTTTTTTTTTT)-3′ 5′-d(AAAAAAAAAAAAAA)-3′ 235′-(AAAAAAAAAAAAAA)-3′ 16 5′-d(TTTTTTTTTTTTTT)-3′5′-d(AAAAAAAAAAAAAA)-3′ <10 5′-(AAAAAAAAAAAAAA)-3′ 425′-(AAAAAAGAAAAAAA)-3′ 37 5′-d(GTFATATGC)-3′ 5′-d(GCATATCAC)-3′ 265′-(GCAUAUCAC)-3′ 27

[0531] TABLE 3 Monomer X T_(m) T_(m) (° C.) T_(m) (° C.) T_(m) (° C.)Oligo Target No. Na₂HPO₄/EDTA Na₂HPO₄/NaCl/EDTA Na₂HPO₄/TMAC5′-d(TTTTTTTTTTTTTT)-3′ 5′-d(AAAAAAAAAAAAAA)-3′ 235′-(AAAAAAAAAAAAAA)-3′ 23 5′-d(TTTTTTTTTTTTTT)-3′5′-d(AAAAAAAAAAAAAA)-3′ 19 5′-(AAAAAAAAAAAAAA)-3′ 235′-d(TTTTTTTTTTTTTT)-3′ 5′-d(AAAAAAAAAAAAAA)-3′ 9 5′-(AAAAAAAAAAAAAA)-3′15 5′-d(TTTTTTTTTTTTTT)-3′ 5′-d(AAAAAAAAAAAAAA)-3′ 55′-(AAAAAAAAAAAAAA)-3′ 14

[0532] TABLE 4 Monomer Y T_(m) T_(m) (° C.) T_(m) (° C.) T_(m) (° C.)Oligo Target No. Na₂HPO₄/EDTA Na₂HPO₄/NaCl/EDTA Na₂HPO₄/TMAC5′-d(TTTTTTTTTTTTTT)-3′ 5′-d(AAAAAAAAAAAAAA)-3′ 365′-(AAAAAAAAAAAAAA)-3′ 37 5′-d(TTTTTTTTTTTTTT)-3′5′-d(AAAAAAAAAAAAAA)-3′ 35 5′-(AAAAAAAAAAAAAA)-3′ 375′-d(TTTTTTTTTTTTTT)-3′ 5′-d(AAAAAAAAAAAAAA)-3′ 355′-(AAAAAAAAAAAAAA)-3′ 36 5′-d(TTTTTTTTTTTTTT)-3′5′-d(AAAAAAAAAAAAAA)-3′ 32 5′-(AAAAAAAAAAAAAA)-3′ 335′-d(TTTTTTTTTTTTTT)-3′ 5′-d(AAAAAAAAAAAAAA)-3′ 365′-(AAAAAAAAAAAAAA)-3′ 36 5′-d(TTTTTTTTTTTTTT)-3′5′-d(AAAAAAAAAAAAAA)-3′ 58 5′-(AAAAAAAAAAAAAA)-3′ 58 5′-d(GTGATATGC)-3′5′-d(GCATATCAC)-3′ 35 5′-(GCAUAUCAC)-3′ 35

[0533] TABLE 5 Monomer Z T_(m) Melting temperature (T_(m)/° C.) OligoTarget No. Y = A Y = C Y = T Y = G 5′-r(GTGATATGC)-3′ 5′-d(GCATYTCAC)-3′1 55 34 38 37 5′-r(GUGAUAUGC)-3′ 5′-d(GCATYTCAC)-3′ 2 27 <10 <10 <105′-r(GTGATATGC)-3′ 5′-r(GCAUYUCAC)-3′ 3 63 45 — — 5′-r(GUGAUAUGC)-3′5′-r(GCAUYUCAC)-3′ 4 38 22 — —

[0534] TABLE 6 Monomer Z T_(m) Oligo Target No. Melting temperature(T_(m)/° C.) 5′-d(GTGATATGC)-3′ 5′-d(GCATATCAC)-3′ 1 285′-d(GTGATATGC)-3′ 5′-d(GCATATCAC)-3′ 2 44 5′-d(GTGATATGC)-3′5′-d(GCATATCAC)-3′ 3 40 5′-d(GTGATATGC)-3′ 5′-d(GCATATCAC)-3′ 4 635′-r(GTGATATGC)-3′ 5′-d(GCATATCAC)-3′ 5 74 5′-(GTGATATG^(Me)C)-3′5′-d(GCATATCAC)-3′ 6 85

[0535] TABLE 7 Monomer Z (all-phosphoromonothioate oligonucleotides)T_(m) Oligo Target No. Melting temperature (T_(m)/° C.)5′-d(G^(S)T^(S)G^(S)A^(S)T^(S)A^(S)T^(S)G^(S)C)-3′ 5′-d(GCATATCAC)-3′ 121 5′-d(G^(S)T^(S)G^(S)A^(S)T^(S)A^(S)T^(S)G^(S)C)-3′ 5′-r(GCAUAUCAC)-3′2 17 5′-d(G^(S)T^(S)G^(S)A^(S)T^(S)A^(S)T^(S)G^(S)C)-3′5′-d(GCATATCAC)-3′ 3 415′-d(G^(S)T^(S)G^(S)A^(S)T^(S)A^(S)T^(S)G^(S)C)-3′ 5′-r(GCAUAUCAC)-3′ 447

[0536] TABLE 8 Monomer thio-Z (U^(S)) T_(m) Oligo Target No. Meltingtemperature (T_(m)/° C.) 5′-d(GTGAU^(S)ATGC)-3′ 5′-d(GCATATCAC)-3′ 1 345′-d(GTGAU^(S)ATGC)-3′ 5′-(GCAUAUCAC)-3′ 2 365′-d(GU^(S)GAU^(S)AU^(S)GC)-3′ 5′-d(GCATATCAC)-3′ 3 425′-d(GU^(S)GAU^(S)AU^(S)GC)-3′ 5′-(GCAUAUCAC)-3′ 4 52 5′-d(GTGTTTTGC)-3′5′-(GCAAAACAC)-3′ 5 27 5′-d(GU^(S)GU^(S)U^(S)U^(S)U^(S)U^(S)GC)-3′5′-d(GCAAAACAC)-3′ 6 51

[0537] TABLE 9 Monomers amino-Z (T^(NH)) and methylamino-Z (T^(NMe))T_(m) Oligo Target No. Melting temperature (T_(m)/° C.)5′-d(GTGAT^(NH)ATGC)-3′ 5′-d(GCATATCAC)-3′ 1 33 5′-d(GTGAT^(NH)ATGC)-3′5′-(GCAUAUCAG)-3′ 2 34 5′-d(GT^(NH)GAT^(NH)AT^(NH)GC)-3′5′-d(GCATATCAC)-3′ 3 39 5′-d(GT^(NH)GAT^(NH)AT^(NH)GC)-3′5′-(GCAUAUCAC)-3′ 4 47 5′-d(GTGAT^(NMe)ATGC)-3′ 5′-d(GCATATCAC)-3′ 5 335′-d(GTGAT^(NMe)ATGC)-3′ 5′-(GCAUAUCAC)-3′ 6 365′-d(GT^(NMe)GAT^(NMe)AT^(NMe)GC)-3′ 5′-d(GCATATCAC)-3′ 7 395′-d(GT^(NMe)GAT^(NMe)AT^(NMe)GC)-3′ 5′-(GCAUAUCAC)-3′ 8 495′-d(GT^(NMe)GT^(NH)T^(NMe)T^(NH)T^(NMe)GC)-3′ 5′-d(GCAAAACAC)-3′ 9 475′-d(GT^(NMe)GT^(NH)T^(NMe)T^(NH)T^(NMe)GC)-3′ 5′-(GCAAAACAC)-3′ 10 63

[0538] TABLE 10 T9 oligo T16 oligos LNA T9 oligo No oligo Steps A₂₆₀units A₂₆₀ units A₂₆₀ units Control poly (rA) added 5.0/5.0 5.0/5.05.0/5.0 5.0/5.0 poly (rA) breakthrough 1.75/1.61 1.84/1.78 1.83/1.825.09/5.14 ∴poly (rA) bound 3.25/3.39 3.16/3.22 3.17/3.18 0.0/0.0 % poly(rA) bound 65.0%/67.8% 63.2%/64.4% 63.4%/63.6% 0.0%/0.0% Low SaltWash/Elute 0.24/0.24 0.11/0.12 .053/.055 0.14/0.13 TE Elute 15 min RT2.37/2.72 0.83/0.93 0.02/0.04 0.01/0.02 TE Elute O.N. RT 0.38/0.371.76/1.69 0.11/0.07 .003/.004 TE Elute 30 min 65° C. .047/.040 0.38/0.461.62/1.70 .005/.004 10 mM Tris pH 10 Elute .002/.002 0.03/0.03 0.10/0.100.01/0.01  1 mM HCl pH 4.0 Elute 0.07/0.06 0.06/0.04 0.26/0.23 0.01/0.01Ave. A₂₆₀ Recovered 3.20 3.14 2.18 — Ave. % A₂₆₀ Recovered 96.4% 98.4%68.7% —

1. An oligomer (hereinafter termed “LNA modified oligonucleotide”)comprising at least one nucleoside analogue (hereinafter termed “LNA”)of the general formula I

wherein X is selected from —O—, —S—, —N(R^(N*)), —C(R⁶R^(6*))—,—O—C(R⁷R^(7*))—, —C(R⁶R^(6*))—O—, —S—C(R⁷R^(7*))—, —C(R⁶R^(6*))—S—,—N(R^(N*))—C(R⁷R^(7*))—, —C(R⁶R^(6*))—N(R^(N*))—, and—C(R⁶R^(6*))—C(R⁷R^(7*))—; B is selected from hydrogen, hydroxy,optionally substituted C₁₋₄-alkoxy, optionally substituted C₁₋₄-alkyl,optionally substituted C₁₋₄-acyloxy, nucleobases, DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands; P designates the radical positionfor an internucleoside linkage to a succeeding monomer, or a 5′-terminalgroup, such internucleoside linkage or 5′-terminal group optionallyincluding the substituent R⁵; one of the substituents R², R^(2*), R³,and R^(3*) is a group P* which designates an internucleoside linkage toa preceding monomer, or a 3′-terminal group; one or two pairs ofnon-geminal substituents selected from the present substituents ofR^(1*), R^(4*), R⁵, R^(5*), R⁶, R^(6*), R⁷, R^(7*), R^(N*), and the onesof R², R^(2*), R³, and R^(3*) not designating P* each designates abiradical consisting of 1-8 groups/atoms selected from —C(R^(a)R^(b))—,—C(R^(a))═C(R^(a))—, —C(R^(a))═N—, —O—, —Si(R^(a))₂—, —S—, —SO₂—,—N(R^(a))—, and >C=Z, wherein Z is selected from —O—, —S—, and—N(R^(a))—, and R^(a) and R^(b) each is independently selected fromhydrogen, optionally substituted C₁₋₁₂-alkyl, optionally substitutedC₂₋₂-alkenyl, optionally substituted C₂₋₁₂-alkynyl, hydroxy,C₁₋₁₂-alkoxy, C₂₋₁₂-alkenyloxy, carboxy, C₁₋₁₂-alkoxycarbonyl,C₁₋₁₂-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy,arylcarbonyl, heteroaryl, heteroaryloxycarbonyl, heteroaryloxy,heteroarylcarbonyl, amino, mono- and di(C₁₋₁₆-alkyl)amino, carbamoyl,mono- and di(C₁₋₆-alkyl)-amino-carbonyl, amino-C₁₋₆-alkyl-aminocarbonyl,mono- and di(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl,C₁₋₆-alkyl-carbonylamino, carbamido, C₁₋₆-alkanoyloxy, sulphono,C₁₋₆-alkylsulphonyloxy, nitro, azido, sulphanyl, C₁₋₆-alkylthio,halogen, DNA intercalators, photochemically active groups,thermochemically active groups, chelating groups, reporter groups, andligands, where aryl and heteroaryl may be optionally substituted, andwhere two geminal substituents R^(a) and R^(b) together may designateoptionally substituted methylene (═CH₂), and wherein two non-geminal orgeminal substitutents selected from R^(a), R^(b), and any of thesubstituents R^(1*), R², R^(2*), R³, R^(3*), R^(4*), R⁵, R^(5*), R⁶ andR^(6*), R⁷, and R^(7*) which are present and not involved in P, P or thebiradical(s) together may form an associated biradical selected frombiradicals of the same kind as defined before; said pair(s) ofnon-geminal substituents thereby forming a mono- or bicyclic entitytogether with (i) the atoms to which said non-geminal substituents arebound and (ii) any intervening atoms; and each of the substituentsR^(1*), R², R^(2*), R³, R^(4*), R⁵, R^(5*), R⁶ and R^(6*), R⁷, andR^(7*) which are present and not involved in P, P* or the biradical(s),is independently selected from hydrogen, optionally substitutedC₁₋₁₂-alkyl, optionally substituted C₂₋₁₂-alkenyl, optionallysubstituted C₂₋₁₂-alkynyl, hydroxy, C₁₋₁₂-alkoxy, C₂₋₁₂-alkenyloxy,carboxy, C₁₋₁₂-alkoxycarbonyl, C₁₋₁₂-alkylcarbonyl, formyl, aryl,aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di(C₁₋₆-alkyl)amino, carbamoyl, mono- anddi(C₁₋₆-alkyl)-aminocarbonyl, amino-C₁₋₆-alkyl-aminocarbonyl, mono- anddi(C₁₋₆-alkyl)amino-C₁₋₆-alkylaminocarbonyl, C₁₋₆-alkyl-carbonylamino,carbamido, C₁₋₆-alkanoyloxy, sulphono, C₁₋₆-alkylsulphonyloxy, nitro,azido, sulphanyl, C₁₋₆-alkylthio, halogen, DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands, where aryl and heteroaryl may beoptionally substituted, and where two geminal substituents together maydesignate oxo, thioxo, imino, or optionally substituted methylene, ortogether may form a spiro biradical consisting of a 1-5 carbon atom(s)alkylene chain which is optionally interrupted and/or terminated by oneor more heteroatoms/groups selected from —O—, —S—, and —(NR^(N))— whereR^(N) is selected from hydrogen and C₁₋₄-alkyl, and where two adjacent(non-geminal) substituents may designate an additional bond resulting ina double bond; and R^(N*), when present and not involved in a biradical,is selected from hydrogen and C₁₋₄-alkyl; and basic salts and acidaddition salts thereof; with the proviso that, (i) R³ and R⁵ do nottogether designate a biradical selected from —CH₂—CH₂—, —O—CH₂—, whenLNA is a bicyclic nucleoside analogue; (ii) R³, R⁵, and R⁵* do nottogether designate a triradical —CH₂—CH(−)—CH₂— when LNA is a tricyclicnucleoside analogue; (iii) R^(1*) and R^(6*) do not together designate abiradical —CH₂— when LNA is a bicyclic nucleoside analogue; and (iv)R^(4*) and R^(6*) do not together designate a biradical —CH₂— when LNAis a bicyclic nucleoside analogue.
 2. An oligomer according to claim 1,wherein the one or two pairs of non-geminal substituents, constitutingone or two biradical(s), respectively, are selected from the presentsubstituents of R^(1*), R^(4*), R⁶, R^(6*), R⁷, R^(7*), R^(N*), and theones of R², R^(2*), R³, and R^(3*) not designating P*.
 3. An oligomeraccording to claim 1, comprising 1-10000 LNA(s) of the general formula Iand 0-10000 nucleosides selected from naturally occurring nucleosidesand nucleoside analogues, with the proviso that the sum of the number ofnucleosides and the number of LNA(s) is at least
 2. 4. An oligomeraccording to claim 3, wherein at least one LNA comprises a nucleobase asthe substituent B.
 5. An oligomer according to claim 1, wherein one ofthe substituents R³ and R^(3*) designates P*.
 6. An oligomer accordingto claim 1, wherein the LNA(s) has/have the following formula Ia

wherein P, P*, B, X, R^(1*), R², R^(2*), R³, R^(4*), R⁵, and R^(5*) areas defined in claim
 1. 7. An oligomer according to claim 6, whereinR^(3*) designates P*.
 8. An oligomer according to claim 1, comprisingone biradical constituted by a pair of (two) non-geminal substituents.9. An oligomer according to claim 1, wherein X is selected from—(CR⁶R^(6*))—, —O—, —S—, and —N(R^(N*))—.
 10. An oligomer according toclaim 1, wherein the biradical(s) constituted by pair(s) of non-geminalsubstituents is/are selected from —(CR*R*)_(r)—Y—(CR*R*)_(s)—,—(CR*R*)_(r)—Y—(CR*R*)_(s)—Y—, —Y—(CR*R*)_(r+s)—Y—,—Y—(CR*R*)_(r)—Y—(CR*R*)_(s)—, —(CR*R*)_(r+s)—, —Y—, —Y—Y—, wherein eachY is independently selected from —O—, —S—, —Si(R*)₂—, —N(R*)—, >C═O,—C(═O)—N(R*)—, and —N(R*)—C(═O)—, each R* is independently selected fromhydrogen, halogen, azido, cyano, nitro, hydroxy, mercapto, amino, mono-or di(C₁₋₆-alkyl)amino, optionally substituted C₁₋₆-alkoxy, optionallysubstituted C₁₋₆-alkyl, DNA intercalators, photochemically activegroups, thermochemically active groups, chelating groups, reportergroups, and ligands, and/or two adjacent (non-geminal) R* may togetherdesignate a double bond, and each of r and s is 0-4 with the provisothat the sum r+s is 1-5.
 11. An oligomer according to claim 10, whereineach biradical is independently selected from —Y—, —(CR*R*)_(r+s)—,—(CR*R*)_(r)—Y—(CR*R*)_(s)—, and —Y—(CR*R*)_(r+s)—Y—, wherein and eachof r and s is 0-3 with the proviso that the sum r+s is 1-4.
 12. Anoligomer according to claim 11, wherein (i) R^(2*) and R^(4*) togetherdesignate a biradical selected from —Y—, —(CR*R*)_(r+s+1)—,—(CR*R*)_(r)—Y—(CR*R*)_(s)—, and —Y—(CR*R*)_(r+s)—Y—; (ii) R² and R³together designate a biradical selected from —Y—, —(CR*R*)_(r+s)—,—(CR*R*)_(r)—Y—(CR*R*)_(s)—, and —Y—(CR*R*)_(r+s)—Y—; (iii) R^(2*) andR³ together designate a biradical selected from —Y—, —(CR*R*)_(r+s)—,—(CR*R*)_(r)—Y—(CR*R*)_(s)—, and —Y—(CR*R*)_(r+s)—Y—; (iv) R³ and R⁴together designate a biradical selected from —Y—, —(CR*R*)_(r+s)—,—(CR*R*)_(r)—Y—(CR*R*)_(s)—, and —Y—(CR*R*)_(r+s)—Y—; (v) R³ and R⁵together designate a biradical selected from —Y′—, —(CR*R*)_(r+s+1)—,—(CR*R*)_(r)—Y—(CR*R*)_(s)—, and —Y—(CR*R*)_(r+s)—Y—; (vi) R^(1*) andR^(4*) together designate a biradical selected from —Y′—,—(CR*R*)_(r+s+1)—, —(CR*R*)_(r)—Y—(CR*R*)_(s)—, and—Y—(CR*R*)_(r+s)—NR*—; or (vii) R^(1*) and R^(2*) together designate abiradical selected from —Y—, —(CR*R*)_(r+s)—,—(CR*R*)_(r)—Y—(CR*R*)_(s)—, and —Y—(CR*R*)_(r+s)—Y—; wherein each of rand s is 0-3 with the proviso that the sum r+s is 1-4, and where Y′ isselected from —NR*—C(═O)— and —C(═O)—NR*—.
 13. An oligomer according toclaim 12, wherein one of the following criteria applies for at least oneLNA: (i) R^(2*) and R^(4*) together designate a biradical selected from—O—, —S—, —N(R*)—, —(CR*R*)_(r+s+1)—, —(CR*R*)_(r)—O—(CR*R*)_(s)—,—(CR*R*)_(r)—O—(CR*R*)_(s)—, —(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—,—O—(CR*R*)_(r+s)—O—, —S—(CR*R*)_(r+s)—O—, —O—(CR*R*)_(r+s)—S—,—N(R*)—(CR*R*)_(r+s)—O—, —O—(CR*R*)_(r+s)—N(R*)—, —S—(CR*R*)_(r+s)—S—,—N(R*)—(CR*R*)_(r+s)—N(R*)—, —N(R*)—(CR*R*)_(r+s)—S—, and—S—(CR*R*)_(r+s)—N(R*)—; (ii) R² and R³ together designate a biradicalselected from —O—, —(CR*R*)_(r+s)—, —(CR*R*)_(r)—O—(CR*R*)_(s)—,—(CR*R*)_(r)—S—(CR*R*)_(s)—, and —(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—; (iii)R^(2*) and R³ together designate a biradical selected from —O—,—(CR*R*)_(r+s)—, —(CR*R*)_(r)—O—(CR*R*)_(s)—,—(CR*R*)_(r)—S—(CR*R*)_(s)—, and —(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—; (iv)R³ and R^(4*) together designate a biradical selected from—(CR*R*)_(r)—O—(CR*R*)_(s)—, —(CR*R*)_(r)—S—(CR*R*)_(s)—, and—(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—; (v) R³ and R⁵ together designate abiradical selected from —(CR*R*)_(r)—O—(CR*R*)_(s)—,—(CR*R*)_(r)—S—(CR*R*)_(s)—, and —(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—; or(vi) R^(1*) and R^(4*) together designate a biradical selected from—(CR*R*)_(r)—O—(CR*R*)_(s)—, —(CR*R*)_(r)—S—(CR*R*)_(s)—, and—(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—; (vii) R^(1*) and R^(2*) togetherdesignate a biradical selected from —(CR*R*)_(r)—O—(CR*R*)_(s)—,—(CR*R*)_(r)—S—(CR*R*)_(s)—, and —(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—;wherein each of r and s is 0-3 with the proviso that the sum r+s is 1-4,and where X is selected from —O—, —S—, and —N(R^(H))— where R^(H)designates hydrogen or C₁₋₄-alkyl.
 14. An oligomer according to claim13, wherein R^(3*) designates P*.
 15. An oligomer according to claim 14,wherein R^(2*) and R^(4*) together designate a biradical.
 16. Anoligomer according to claim 15, wherein X is O, R² is selected fromhydrogen, hydroxy, and optionally substituted C₁₋₆-alkoxy, and R^(1*),R³, R⁵, and R^(5*) designate hydrogen.
 17. An oligomer according toclaim 16, wherein the biradical is selected from —O—,—(CH₂)₀₋₁—O—(CH₂)₁₋₃—, —(CH₂)₀₋₁—S—(CH₂)₁₋₃—, and—(CH₂)₀₋₁—N(R^(N))—(CH₂)₁₋₃—.
 18. An oligomer according to claim 17,wherein the biradical is selected from —O—CH₂—, —S—CH₂— and—N(R^(N))—CH₂—. 19 An oligomer according to claim 15, wherein B isselected from nucleobases.
 20. An oligomer according to claim 19,wherein the oligomer comprises at least one LNA wherein B is selectedfrom adenine and guanine and at least one LNA wherein B is selected fromthymine, cytosine and urasil.
 21. An oligomer according to claim 16,wherein the biradical is —(CH₂)₂₋₄—.
 22. An oligomer according to claim14, wherein R² and R³ together designate a biradical.
 23. An oligomeraccording to claim 22, wherein X is O, R^(2*) is selected from hydrogen,hydroxy, and optionally substituted C₁₋₆-alkoxy, and R^(1*), R^(4*), R⁵,and R^(5*) designate hydrogen.
 24. An oligomer according to claim 23,wherein the biradical is —(CH₂)₀₋₁—O—(CH₂)₁₋₃—.
 25. An oligomeraccording to claim 23, wherein the biradical is —(CH₂)₁₋₄—.
 26. Anoligomer according to claim 14, wherein one R* is selected fromhydrogen, hydroxy, optionally substituted C₁₋₆-alkoxy, optionallysubstituted C₁₋₆-alkyl, DNA intercalators, photochemically activegroups, thermochemically active groups, chelating groups, reportergroups, and ligands, and any remaining substituents R* are hydrogen. 27.An oligomer according to claim 14, wherein a group R* in the biradicalof at least one LNA is selected from DNA intercalators, photochemicallyactive groups, thermochemically active groups, chelating groups,reporter groups, and ligands.
 28. An oligomer according to claim 14,wherein the LNA(s) has/have the general formula Ia.
 29. An oligomeraccording to claim 1 of the general formula Ia

wherein X is —O—; B is selected from nucleobases, DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands; P designates the radical positionfor an internucleoside linkage to a succeeding monomer, or a 5′-terminalgroup, such internucleoside linkage or 5′-terminal group optionallyincluding the substituent R⁵; R^(3*) is a group P* which designates aninternucleoside linkage to a preceding monomer, or a 3′-terminal group;R^(2*) and R^(4*) together designate a biradical selected from —O—, —S—,—N(R*)—, —(CR*R*)_(r+s+1)—, —(CR*R*)_(r)—O—(CR*R*)_(s)—,—(CR*R*)_(r)—S—(CR*R*)_(s)—, —(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—,—O—(CR*R*)_(r+s)—O—, —S—(CR*R*)_(r+s)—O—, —O—(CR*R*)_(r+s)—S—,—N(R*)—(CR*R*)_(r+s)—O—, —O—(CR*R*)_(r+s)—N(R*)—, —S—(CR*R*)_(r+s)—S—,—N(R*)—(CR*R*)_(r+s)—N(R*)—, —N(R*)—(CR*R*)_(r+s)—S—, and—S—(CR*R*)_(r+s)—N(R*)—; wherein each R* is independently selected fromhydrogen, halogen, azido, cyano, nitro, hydroxy, mercapto, amino, mono-or di(C₁₋₆-alkyl)amino, optionally substituted C₁₋₆-alkoxy, optionallysubstituted C₁₋₆-alkyl, DNA intercalators, photochemically activegroups, thermochemically active groups, chelating groups, reportergroups, and ligands, and/or two adjacent (non-geminal) R may togetherdesignate a double bond, and each of r and s is 0-3 with the provisothat the sum r+s is 1-4; each of the substituents R^(1*), R², R³, R⁵,and R^(5*) is independently selected from hydrogen, optionallysubstituted C₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl, hydroxy,C₁₋₆-alkoxy, C₂₋₆-alkenyloxy, carboxy, C₁₋₆-alkoxycarbonyl,C₁₋₆-alkylcarbonyl, formyl, amino, mono- and di(C₁₋₆-alkyl)amino,carbamoyl, mono- and di(C₁₋₆-alkyl)-aminocarbonyl,C₁₋₆-alkyl-carbonylamino, carbamido, azido, C₁₋₆-alkanoyloxy, sulphono,sulphanyl, C₁₋₆-alkylthio, DNA intercalators, photochemically activegroups, thermochemically active groups, chelating groups, reportergroups, and ligands, and halogen, where two geminal substituentstogether may designate oxo; and basic salts and acid addition saltsthereof.
 30. An oligomer according to claim 29, wherein one R* isselected from hydrogen, hydroxy, optionally substituted C₁₋₆-alkoxy,optionally substituted C₁₋₆-alkyl, DNA intercalators, photochemicallyactive groups, thermochemically active groups, chelating groups,reporter groups, and ligands, and any remaining substituents R* arehydrogen.
 31. An oligomer according to claim 29, wherein the biradicalis selected from —O—, —(CH₂)₀₋₁—O—(CH₂)₁₋₃—, —(CH₂)₀₋₁—S—(CH₂)₁₋₃—,—(CH₂)O, —N(R^(N))(CH₂)₁₋₃—, and —(CH₂)₂₋₄—.
 32. An oligomer accordingto claim 31, wherein the biradical is selected from —O—CH₂—, —S—CH₂— and—N(R^(N))—CH₂—.
 33. An oligomer according to claim 29, wherein B isselected from nucleobases.
 34. An oligomer according to claim 33,wherein the oligomer comprises at least one LNA wherein B is selectedfrom adenine and guanine and at least one LNA wherein B is selected fromthymine, cytosine and urasil.
 35. An oligomer according to claim 29,wherein R² is selected from hydrogen, hydroxy and optionally substitutedC₁₋₆-alkoxy, and R^(1*), R³, R⁵, and R⁵ designate hydrogen.
 36. Anoligomer according to claim 1, wherein any internucleoside linkage ofthe LNA(s) is selected from linkages consisting of 2 to 4 groups/atomsselected from —CH₂—, —O—, —S—, —NR^(H)—, >C═O, >C═NR^(H), >C═S,—Si(R″)₂— —SO—, —S(O)₂—, —P(O)₂—, —P(O,S)—, —P(S)₂—, —PO(R″)—,—PO(OCH₃)—, and —PO(NHR^(H))—, where R^(H) is selected form hydrogen andC₁₋₄-alkyl, and R″ is selected from C₁₋₆-alkyl and phenyl.
 37. Anoligomer according to claim 36, wherein any internucleoside linkage ofthe LNA(s) is selected from —CH₂—CH₂—CH₂—, —CH₂—CO—CH₂—, —CH₂—CHOH—CH₂—,—O—CH₂—O—, —O—CH₂—CH₂—, —O—CH₂—CH═, —CH₂—CH₂—O—, —NR^(H)—CH₂—CH₂—,—CH₂—CH₂—NR^(H), CH₂NR^(H)—, CH₂—, —O—CH₂—CH₂—NR^(H)—, —NR^(H)—CO—O—,—NR^(H) CO—NR^(H)—, —NR^(H)CS—NR^(H)—, NR^(H)—C(═NR^(H))—NR^(H)—,—NR^(H)—CO—CH₂—NR^(H)—, —O—CO—O—, —O—CO—CH₂—O—, —O—CH₂—CO—O—,—CH₂—CO—NR^(H)—, —O—CO—NR^(H)—, —NR^(H)—CO—CH₂—, —O—CH₂—CO—NR^(H)—,—O—CH₂—CH₂—NR^(H)—, —CH═N—O—, —CH₂—NR^(H)—O—, —CH₂—O—N═, —CH₂O—NR^(H)—,CO—NR^(H)CH₂—, —CH₂—NR^(H)O—, —CH₂—NR^(H)—CO—, —O—NR^(H)—CH₂—,—O—NR^(H)—, —O—CH₂—S—, —S—CH₂—O—, —CH₂—CH₂—S—, —O—CH₂—CH₂—S—,—S—CH₂—CH═, —S—CH₂—CH₂—, —S—CH₂—CH₂—O—, —S—CH₂—CH₂—S—, —CH₂—S—CH₂—,—CH₂—SO—CH₂—, —CH₂—SO₂—CH₂—, —O—SO—O—, —O—S(O)₂—O—, —O—S(O)₂—CH₂—,—O—S(O)₂—NR^(H), NR^(H)—S(O)₂—CH₂—, —O—S(O)₂—CH₂—, —O—P(O)₂—O—,—O—P(O,S)—O—, —O—P(S)₂—O—, —S—P(O)₂—O—, —S—P(O,S)—O—, —S—P(S)₂—O—,—O—P(O)₂—S—, —O—P(O,S)—S—, —O—P(S)₂—S—, —S—P(O)₂—S—, —S—P(O,S)—S—,—S—P(S)₂—S—, —O—PO(R″)—O—, —O—PO(OCH₃)—O—, —O—PO(BH₃)—O—,—O—PO(NHR^(N))—O—, —O—P(O)₂—NR^(H)—, —NR^(H)—P(O)₂—O—,—O—P(O,NR^(H))—O—, and —O—Si(R″)₂—O—.
 38. An oligomer according to claim37, wherein any internucleoside linkage of the LNA(s) is selected from—CH₂—CO—NR^(H)—, —CH₂—NR^(H)—O—, —S—CH₂—O—, —O—P(O)₂—O—, —O—P(O,S)—O—,—O—P(S)₂—O—, —NR^(H)—P(O)₂—O—, —O—P(O,NR^(H))—O—, —O—PO(R″)—O—,—O—PO(CH₃)—O—, and —O—PO(NHR^(N))—O—, where R^(H) is selected formhydrogen and C₁₋₄-alkyl, and R″ is selected from C₁₋₆-alkyl and phenyl.39. An oligomer according to claim 1, wherein each of the substituentsR^(1*), R², R^(2*), R³, R^(3*), R^(4*), R⁵, R^(5*), R⁶, R^(6*), R⁷, andR^(7*) of the LNA(s), which are present and not involved in P, P* or thebiradical(s), is independently selected from hydrogen, optionallysubstituted C₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl, hydroxy,C₁₋₆-alkoxy, C₂₋₆-alkenyloxy, carboxy, C₁₋₆-alkoxycarbonyl,C₁₋₆-alkylcarbonyl, formyl, amino, mono- and di(C₁₋₆-alkyl)amino,carbamoyl, mono- and di(C₁₋₆-alkyl)-aminocarbonyl,C₁₋₆-alkyl-carbonylamino, carbamido, azido, C₁ r-alkanoyloxy, sulphono,sulphanyl, C₁₋₆-alkylthio, DNA intercalators, photochemically activegroups, thermochemically active groups, chelating groups, reportergroups, and ligands, and halogen, where two geminal substituentstogether may designate oxo, and where R^(N*), when present and notinvolved in a biradical, is selected from hydrogen and C₁₋₄-alkyl. 40.An oligomer according to claim 1, wherein X is selected from —O—, —S—,and —NR^(N*)—, and each of the substituents R^(1*), R², R^(2*), R³,R^(3*), R^(4*), R⁵, R^(5*), R⁶, R^(6*), R⁷, and R^(7*) of the LNA(s),which are present and not involved in P, P or the biradical(s),designate hydrogen.
 41. An oligomer according to claim 1, wherein P is a5′-terminal group selected from hydrogen, hydroxy, optionallysubstituted C₁₋₆-alkyl, optionally substituted C₁₋₆-alkoxy, optionallysubstituted C₁₋₆-alkylcarbonyloxy, optionally substituted aryloxy,monophosphate, diphosphate, triphosphate, and —W-A′, wherein W isselected from ——O—, —S—, and —N(R^(H))— where R^(H) is selected fromhydrogen and C₁₋₆-alkyl, and where A′ is selected from DNAintercalators, photochemically active groups, thermochemically activegroups, chelating groups, reporter groups, and ligands.
 42. An oligomeraccording to claim 1, wherein P* is a 3′-terminal group selected fromhydrogen, hydroxy, optionally substituted C₁₋₆-alkoxy, optionallysubstituted C₁₋₆-alkylcarbonyloxy, optionally substituted aryloxy, and—W-A′, wherein W is selected from —O—, —S—, and —N(R^(H))— where R^(H)is selected from hydrogen and C₁₋₆-alkyl, and where A′ is selected fromDNA intercalators, photochemically active groups, thermochemicallyactive groups, chelating groups, reporter groups, and ligands.
 43. Anoligomer according to claims 1, having the following formula V:G-[Nu-L]_(n(0))-{([LNA-L]_(m(q))-[Nu-L]_(n(q))}_(q)-G*  V wherein q is1-50; each of n(0), . . . , n(q) is independently 0-10000; each of m(1),. . . , m(q) is independently 1-10000; with the proviso that the sum ofn(0), . . . , n(q) and m(1), . . . , m(q) is 2-15000; G designates a5′-terminal group; each Nu independently designates a nucleosideselected from naturally occurring nucleosides and nucleoside analogues;each LNA independently designates a nucleoside analogue; each Lindependently designates an internucleoside linkage between two groupsselected from Nu and LNA, or L together with G* designates a 3′-terminalgroup; and each LNA-L independently designates a nucleoside analogue ofthe general formula I:

wherein the substituents B, P, P*, R^(1*), R², R^(2*), R³, R^(4*), R⁵,and R^(5*), and X are as defined in claim
 1. 44. An oligomer accordingto claim 1, further comprising a PNA mono- or oligomer segment of theformula

wherein B is a defined above for the formula I, AASC designates hydrogenor an amino acid side chain, t is 1-5, and w is 1-50.
 45. An oligomeraccording to claim 1, which has an increased specificity towardscomplementary ssRNA or ssDNA compared to the native oligonucleotide. 46.An oligomer according to claim 1, which has an increased affinitytowards complementary ssRNA or ssDNA compared to the nativeoligonucleotide.
 47. An oligomer according to claim 1, which is capableof binding to a target sequence in a dsDNA or dsRNA molecule by way of“strand displacement” or by triple helix formation.
 48. An oligomeraccording to claim 1, which is more resistant to nucleases than thenative oligonucleotide.
 49. An oligomer according to claim 1, which hasnucleic acid catalytic activity (LNA modified ribozymes).
 50. Anoligomer comprising at least one nucleoside analogue which imparts tothe oligomer a T_(m) with a complementary DNA oligonucleotide which isat least 2.5° C. higher than that of the corresponding unmodifiedreference oligonucleotide which does not comprise any nucleosideanalogue.
 51. An oligomer according to claim 50, wherein the T_(m) is atleast 2.5×N° C. higher, where N is the number of nucleoside analogues.52. An oligomer comprising at least one nucleoside analogue whichimparts to the oligomer a T_(m) with a complementary RNA oligonucleotidewhich is at least 4.0° C. higher than that of the correspondingunmodified reference oligonucleotide which does not comprise anynucleoside analogue.
 53. An oligomer according to claim 52, wherein theT_(m) is at least 4.0×N° C. higher, where N is the number of nucleosideanalogues.
 54. An oligomer according to claim 50 or 52, wherein theoligomer is as defined in claim 1, where the at least one nucleosideanalogue has the formula I where B is a nucleobase.
 55. An oligomeraccording to claim 50, wherein said oligomer, when hybridised with apartially complementary DNA oligonucleotide having one or moremismatches with said oligomer, exhibits a reduction in T_(m), as aresult of said mismatches, which is equal to or greater than thereduction which would be observed with the corresponding unmodifiedreference oligonucleotide which does not comprise any nucleosideanalogues.
 56. An oligomer according to claim 52, wherein said oligomer,when hybridised with a partially complementary RNA oligonucleotidehaving one or more mismatches with said oligomer, exhibits a reductionin T_(m), as a result of said mismatches, which is equal to or greaterthan the reduction which would be observed with the correspondingunmodified reference oligonucleotide which does not comprise anynucleoside analogues.
 57. An oligomer according to claim 50 or 52, whichhas substantially the same sensitivity of T_(m) to the ionic strength ofthe hybridisation buffer as that of the corresponding unmodifiedreference oligonucleotide.
 58. An oligomer according to claim 50 or 52,which is at least 30% modified.
 59. An oligomer according to claim 50 or52, which has substantially higher 3′-exonucleolytic stability than thecorresponding unmodified reference oligonucleotide.
 60. A nucleosideanalogue (hereinafter LNA) of the general formula II

wherein the substituent B is selected from nucleobases, DNAintercalators, photochemically active groups, thermochemically activegroups, chelating groups, reporter groups, and ligands; X is selectedfrom —O—, —S—, —N(R^(N))—, and —C(R⁶R⁶)—; one of the substituents R²,R^(2*), R³, and R^(3*) is a group Q*; each of Q and Q* is independentlyselected from hydrogen, azido, halogen, cyano, nitro, hydroxy, Prot-O—,Act-O—, mercapto, Prot-S—, Act-S—, C₁₋₆-alkylthio, amino,Prot-N(R^(H))—, Act-N(R^(H))—, mono- or di(C₁₋₆-alkyl)amino, optionallysubstituted C₁₋₆-alkoxy, optionally substituted C₁₋₆-alkyl, optionallysubstituted C₂₋₆-alkenyl, optionally substituted C₂₋₆-alkenyloxy,optionally substituted C₂₋₆-alkynyl, optionally substitutedC₂₋₆-alkynyloxy, monophosphate, diphosphate, triphosphate, DNAintercalators, photochemically active groups, thermochemically activegroups, chelating groups, reporter groups, ligands, carboxy, sulphono,hydroxymethyl, Prot-O—CH₂—, Act-O—CH₂—, aminomethyl, Prot-N(R^(H))—CH₂—,Act-N(R^(H))—CH₂—, carboxymethyl, sulphonomethyl, where Prot is aprotection group for —OH, —SH, and —NH(R^(H)), respectively, Act is anactivation group for —OH, —SH, and —NH(R^(H)), respectively, and R^(H)is selected from hydrogen and C₁₋₆-alkyl; (i) R^(2*) and R^(4*) togetherdesignate a biradical selected from —O—, —(CR*R*)_(r+s+1)—,—(CR*R*)_(r)—O—(CR*R*)_(s)—, —(CR*R*)_(r)—S—(CR*R*)_(s)—,—(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—, —O—(CR*R*)_(r+s)—O—,—S—(CR*R*)_(r+s)—O—, —O—(CR*R*)_(r+s)—S—, —N(R*)—(CR*R*)_(r+s)—O—,—O—(CR*R*)_(r+s)—N(R*)—, —S—(CR*R*)_(r+s)—S—,—N(R*)—(CR*R*)_(r+s)—N(R*)—, —N(R*)—(CR*R*)_(r+s)—S—, and—S—(CR*R*)_(r+s)—N(R*)—; (ii) R² and R³ together designate a biradicalselected from —O—, —(CR*R*)_(r+s)—, —(CR*R*)_(r)—O—(CR*R*)_(s)—,—(CR*R*)_(r)—S—(CR*R*)_(s)—, and —(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—; (iii)R^(2*) and R³ together designate a biradical selected from —O—,—(CR*R*)_(r+s)—, —(CR*R*)_(r)—O—(CR*R*)_(s)—,—(CR*R*)_(r)—S—(CR*R*)_(s)—, and —(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—; (iv)R³ and R^(4*) together designate a biradical selected from—(CR*R*)_(r)—O—(CR*R*)_(s)—, —(CR*R*)_(r)—S—(CR*R*)_(s)—, and—(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—; (v) R³ and R⁵ together designate abiradical selected from —(CR*R*)_(r)—O—(CR*R*)_(s)—,—(CR*R*)_(r)—S—(CR*R*)_(s)—, and —(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—; or(vi) R^(1*) and R^(2*) together designate a biradical selected from—(CR*R*)_(r)—O—(CR*R*)_(s)—, —(CR*R*)_(r)—S—(CR*R*)_(s)—, and—(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—; (vii) R^(1*) and R^(2*) togetherdesignate a biradical selected from —(CR*R*)_(r)—O—(CR*R*)_(s)—,—(CR*R*)_(r)—S—(CR*R*)_(s)—, and —(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—;wherein each R* is independently selected from hydrogen, halogen, azido,cyano, nitro, hydroxy, mercapto, amino, mono- or di(C₁₋₆-alkyl)amino,optionally substituted C₁₋₆-alkoxy, optionally substituted C₁₋₆-alkyl,DNA intercalators, photochemically active groups, thermochemicallyactive groups, chelating groups, reporter groups, and ligands, and/ortwo adjacent (non-geminal) R* may together designate a double bond, andeach of r and s is 0-3 with the proviso that the sum r+s is 1-4; each ofthe substituents R^(1*), R², R^(2*), R³, R^(4*), R⁵, and R^(5*), whichare not involved in Q, Q* or the biradical, is independently selectedfrom hydrogen, optionally substituted C₁₋₁₂-alkyl, optionallysubstituted C₂₋₁₂-alkenyl, optionally substituted C₂₋₁₂-alkynyl,hydroxy, C₁₋₁₂-alkoxy, C₂₋₁₂-alkenyloxy, carboxy, C₁₋₁₂-alkoxycarbonyl,C₁₋₁₂-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy,arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy,heteroarylcarbonyl, amino, mono- and di(C₁₋₆-alkyl)amino, carbamoyl,mono- and di(C₁₋₆-alkyl)-amino-carbonyl, amino-C₁₋₆-alkylaminocarbonyl,mono- and di(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl,C₁₋₆-alkylcarbonylamino, carbamido, C₁₋₆-alkanoyloxy, sulphono,C₁₋₆-alkylsulphonyloxy, nitro, azido, sulphanyl, C₁₋₆-alkylthio,halogen, DNA intercalators, photochemically active groups,thermochemically active groups, chelating groups, reporter groups, andligands, where aryl and heteroaryl may be optionally substituted, andwhere two geminal substituents together may designate oxo, thioxo,imino, or optionally substituted methylene, or together may form a spirobiradical consisting of a 1-5 carbon atom(s) alkylene chain which isoptionally interrupted and/or terminated by one or moreheteroatoms/groups selected from —O—, —S—, and —(NR^(N))— where R^(N) isselected from hydrogen and C₁₋₄-alkyl, and where two adjacent(non-geminal) substituents may designate an additional bond resulting ina double bond; and R^(N), when present and not involved in a biradical,is selected from hydrogen and C₁₋₄-alkyl; and basic salts and acidaddition salts thereof; with the first proviso that, (i) R³ and R⁵ donot together designate a biradical selected from —CH₂—CH₂—, —O—CH₂—, and—O—Si(^(i)Pr)₂—O—Si(^(i)Pr)₂—O—; and with the second proviso that anychemical group (including any nucleobase), which is reactive under theconditions prevailing in oligonucleotide synthesis, is optionallyfunctional group protected.
 61. A nucleoside analogue according to claim60, wherein the group B is selected from nucleobases and functionalgroup protected nucleobases.
 62. A nucleoside analogue according toclaim 60, wherein X is selected from —O—, —S—, and —N(R^(N*))—.
 63. Anucleoside analogue according to claim 60, wherein each of thesubstituents R^(1*), R², R^(2*), R³, R^(3*), R^(4*), R⁵, and R^(5*),which are present and not involved in Q, Q* or the biradical, isindependently selected from hydrogen, optionally substituted C₁₋₆-alkyl,optionally substituted C₂₋₆-alkenyl, hydroxy, C₁₋₆-alkoxy,C₂₋₆-alkenyloxy, carboxy, C₁₋₆-alkoxycarbonyl, C₁₋₆-alkylcarbonyl,formyl, amino, mono- and di(C₁₋₆-alkyl)amino, carbamoyl, mono- anddi(C₁₋₆-alkyl)-amino-carbonyl, C₁₋₆-alkylcarbonylamino, carbamido,azido, C₁₋₆-alkanoyloxy, sulphono, sulphanyl, C₁₋₆-alkylthio, DNAintercalators, photochemically active groups, thermochemically activegroups, chelating groups, reporter groups, ligands, and halogen, wheretwo geminal substituents together may designate oxo, and where R^(N*),when present and not involved in a biradical, is selected from hydrogenand C₁₋₄-alkyl, with the proviso that any hydroxy, amino,mono(C₁₋₆-alkyl)amino, sulfanyl, and carboxy is optionally protected.64. A nucleotide analogue according to claim 60, each of thesubstituents R^(1*), R², R^(2*), R³, R^(3*), R^(4*), and R⁵, R^(5*), R⁶,R^(6*), which are present and not involved in Q* or the biradical,designate hydrogen.
 65. A nucleoside analogue according to claim 60,wherein R^(3*) designates P*.
 66. A nucleoside analogue according toclaims 60, wherein Q is independently selected from hydrogen, azido,halogen, cyano, nitro, hydroxy, Prot-O—, mercapto, Prot-S—,C₁₋₆-alkylthio, amino, Prot-N(R^(H))—, mono- or di(C₁₋₆-alkyl)amino,optionally substituted C₁₋₆-alkoxy, optionally substituted C₁₋₆-alkyl,optionally substituted C₂₋₆-alkenyl, optionally substitutedC₂₋₆-alkenyloxy, optionally substituted C₂₋₆-alkynyl, optionallysubstituted C₂₋₆-alkynyloxy, monophosphate, diphosphate, triphosphate,DNA intercalators, photochemically active groups, thermochemicallyactive groups, chelating groups, reporter groups, ligands, carboxy,sulphono, hydroxymethyl, Prot-O—CH₂—, aminomethyl, Prot-N(R^(H))—CH₂—,carboxymethyl, sulphonomethyl, where Prot is a protection group for —OH,—SH, and —NH(R^(H)), respectively, and R^(H) is selected from hydrogenand C₁₋₆-alkyl; and Q* is selected from hydrogen, azido, halogen, cyano,nitro, hydroxy, Act-O—, mercapto, Act-S—, C₁₋₆-alkylthio, amino,Act-N(R^(H))—, mono- or di(C₁₋₆-alkyl)amino, optionally substitutedC₁₋₆-alkoxy, optionally substituted C₁₋₆-alkyl, optionally substitutedC₂₋₆-alkenyl, optionally substituted C₂₋₆-alkenyloxy, optionallysubstituted C₂₋₆-alkynyl, optionally substituted C₂₋₆-alkynyloxy, DNAintercalators, photochemically active groups, thermochemically activegroups, chelating groups, reporter groups, ligands, carboxy, sulphono,where Act is an activation group for —OH, —SH, and —NH(R^(H)),respectively, and R^(H) is selected from hydrogen and C₁₋₆-alkyl.
 67. Anucleotide analogue according to claim 60, having the general formulaIIa

wherein the substituents Q, B, R^(1*), R², R^(2*), R³, R^(3*), R^(4*),R⁵, and R^(5*) are as defined in claims
 60. 68. A nucleoside analogueaccording to claim 67, wherein R^(3*) designates P*.
 69. A nucleosideanalogue according to claim 68, wherein R^(2*) and R^(4*) togetherdesignate a biradical.
 70. A nucleoside analogue according to claim 69,wherein X is O, R² selected from hydrogen, hydroxy, and optionallysubstituted C₁₋₆-alkoxy, and R^(1*), R³, R⁵, and R^(5*) designatehydrogen.
 71. A nucleoside analogue according to claim 70, wherein thebiradical is selected from —O—, —(CH₂)₀₋₁—O—(CH₂)₁₋₃—,—(CH₂)₀₋₁—S—(CH₂)₁₋₃—, and —(CH₂)₀₋₁—N(R^(N))—(CH₂)₁₋₃—.
 72. Anucleoside analogue according to claim 71, wherein the biradical isselected from —O—CH₂—, —S—CH₂— and —N(R^(N))—CH₂—.
 73. A nucleosideanalogue according to claim 69, wherein B is selected from nucleobases.74. A nucleoside analogue according to claim 73, wherein the oligomercomprises at least one LNA wherein B is selected from adenine andguanine and at least one LNA wherein B is selected from thymine,cytosine and urasil.
 75. A nucleoside analogue according to claim 70,wherein the biradical is —(CH₂)₂₋₄—.
 76. A nucleoside analogue accordingto claim 68, wherein R² and R³ together designate a biradical.
 77. Anucleoside analogue according to claim 76, wherein X is O, R^(2*) isselected from hydrogen, hydroxy, and optionally substituted C₁₋₆-alkoxy,and R^(1*), R^(4*), R⁵, and R^(5*) designate hydrogen.
 78. A nucleosideanalogue according to claim 77, wherein the biradical is—(CH₂)₀₋₁—O—(CH₂)₁₋₃—.
 79. A nucleoside analogue according to claim 77,wherein the biradical is —(CH₂)₁₋₄—.
 80. A nucleoside analogue accordingto claim 68, wherein one R* is selected from hydrogen, hydroxy,optionally substituted C₁₋₆-alkoxy, optionally substituted C₁₋₆-alkyl,DNA intercalators, photochemically active groups, thermochemicallyactive groups, chelating groups, reporter groups, and ligands, and anyremaining substituents R are hydrogen.
 81. A nucleoside analogueaccording to claim 68, wherein a group R* in the biradical of at leastone LNA is selected from DNA intercalators, photochemically activegroups, thermochemically active groups, chelating groups, reportergroups, and ligands.
 82. A nucleoside analogue according to claim 68,wherein the LNA(s) has/have the general formula Ia.
 83. A nucleosideanalogue according to claim 60 of the general formula IIa

wherein X is —O—; B is selected from nucleobases, DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands; R^(3*) is a group Q*; each of Qand Q* is independently selected from hydrogen, azido, halogen, cyano,nitro, hydroxy, Prot-O—, Act-O—, mercapto, Prot-S—, Act-S—,C₁₋₆-alkylthio, amino, Prot-N(R^(H))—, Act-N(R^(H))—, mono- ordi(C₁₋₆-alkyl)amino, optionally substituted C₁₋₆-alkoxy, optionallysubstituted C₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl, optionallysubstituted C₂₋₆-alkenyloxy, optionally substituted C₂₋₆-alkynyl,optionally substituted C₂₋₆-alkynyloxy, monophosphate, diphosphate,triphosphate, DNA intercalators, photochemically active groups,thermochemically active groups, chelating groups, reporter groups,ligands, carboxy, sulphono, hydroxymethyl, Prot-O—CH₂—, Act-O—CH₂—,aminomethyl, Prot-N(R^(H))—CH₂—, Act-N(R^(H))—CH₂—, carboxymethyl,sulphonomethyl, where Prot is a protection group for —OH, —SH, and—NH(R^(H)), respectively, Act is an activation group for —OH, —SH, and—NH(R^(H)), respectively, and R^(H) is selected from hydrogen andC₁₋₆-alkyl; R^(2*) and R^(4*) together designate a biradical selectedfrom —O—, —S, —N(R*)—, —(CR*R*)_(r+s+1)—, —(CR*R*)_(r)—O—(CR*R*)_(s)—,—(CR*R*)_(r)—S—(CR*R*)_(s)—,—(CR*R*)_(r)—N(R*)—(CR*R*)_(s)—,—O—(CR*R*)_(r+s)—O—,—S—(CR*R*)_(r+s)—O—, —O—(CR*R*)_(r+s)—N(R*)—, —N(R*)—(CR*R*)_(r+s)—O—,—O—(CR*R*)_(r+s)—N(R*)—, —S—(CR*R*)_(r+s)—S—,—N(R*)—(CR*R*)_(r+s)—N(R*)—, —N(R*)—(CR*R*)_(r+s)—S—, and—S—(CR*R*)_(r+s)—N(R*)—; wherein each R* is independently selected fromhydrogen, halogen, azido, cyano, nitro, hydroxy, mercapto, amino, mono-or di(C₁₋₆-alkyl)amino, optionally substituted C₁₋₆-alkoxy, optionallysubstituted C₁₋₆-alkyl, DNA intercalators, photochemically activegroups, thermochemically active groups, chelating groups, reportergroups, and ligands, and/or two adjacent (non-geminal) R* may togetherdesignate a double bond, and each of r and s is 0-3 with the provisothat the sum r+s is 1-4; each of the substituents R^(1*), R², R³, R⁵,and R^(5*) is independently selected from hydrogen, optionallysubstituted C₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl, hydroxy,C₁₋₆-alkoxy, C₂₋₆-alkenyloxy, carboxy, C₁₋₆-alkoxycarbonyl,C₁₋₆-alkylcarbonyl, formyl, amino, mono- and di(C₁₋₆-alkyl)amino,carbamoyl, mono- and di(C₁₋₆-alkyl)-aminocarbonyl,C₁₋₆-(alkyl-carbonylamino, carbamido, azido, C₁₋₆-alkanoyloxy, sulphono,sulphanyl, C₁₋₆-alkylthio, DNA intercalators, photochemically activegroups, thermochemically active groups, chelating groups, reportergroups, and ligands, and halogen, where two geminal substituentstogether may designate oxo; and basic salts and acid addition saltsthereof; and with the proviso that any chemical group (including anynucleobase), which is reactive under the conditions prevailing inoligonucleotide synthesis, is optionally functional group protected. 84.A nucleotide analogue according to claim 83, wherein one R* is selectedfrom hydrogen, hydroxy, optionally substituted C₁₋₆-alkoxy, optionallysubstituted C₁₋₆-alkyl, DNA intercalators, photochemically activegroups, thermochemically active groups, chelating groups, reportergroups, and ligands, and any remaining substituents R* are hydrogen. 85.A nucleotide analogue according to claim 83, wherein the biradical isselected from —O—, —(CH₂)₀₋₁—O—(CH₂)₁₋₃—, —(CH₂)₀₋₁—S—(CH₂)₁₋₃—,—(CH₂)₀₋₁—N(R^(N))—(CH₂)₁₋₃—, and —(CH₂)₂₋₄—.
 86. A nucleoside analogueaccording to claim 85, wherein the biradical is selected from —O—CH₂—,—S—CH₂— and —N(R^(N))—CH₂—.
 87. A nucleoside analogue according to claim83, wherein B is selected from nucleobases.
 88. A nucleoside analogueaccording to claim 87, wherein the oligomer comprises at least one LNAwherein B is selected from adenine and guanine and at least one LNAwherein B is selected from thymine, cytosine and urasil.
 89. Anucleoside analogue according to claim 83, wherein B designates anucleobase, X is —O—, R^(2*) and R^(4*) together designate a biradicalselected from —(CH₂)₀₋₁—O—(CH₂)₁₋₃—, —(CH₂)₀₋₁—S—(CH₂)₁₋₃—, and—(CH₂)₀₋₁—N(R^(N))—(CH₂)₁₋₃— where R^(N) is selected from hydrogen andC₁₋₄-alkyl, Q designates Prot-O—, R^(3*) is Q* which designates Act-OH,and R^(1*), R², R³, R⁵, and R^(5*) each designate hydrogen, wherein Actand Prot are as defined in claim
 58. 90. A nucleoside analogue accordingto claim 83, wherein B designates a nucleobase, X is —O—, R^(2*) andR^(4*) together designate a biradical selected from—(CH₂)₀₋₁—O—(CH₂)₁₋₃—, —(CH₂)₀₋₁—S—(CH₂)₁₋₃—, and—(CH₂)₀₋₁N(R^(N))—(CH₂)₁₋₃— where R^(N) is selected from hydrogen andC₁₋₄-alkyl, Q is selected from hydroxy, mercapto, C₁₋₆-alkylthio, amino,mono- or di(C₁₋₆-alkyl)amino, optionally substituted C₁₋₆-alkoxy,optionally substituted C₂₋₆-alkenyloxy, optionally substitutedC₂₋₆-alkynyloxy, monophosphate, diphosphate, and triphosphate, R^(3*) isQ* which is selected from hydrogen, azido, halogen, cyano, nitro,hydroxy, mercapto, C₁₋₆-alkylthio, amino, mono- or di(C₁₋₆-alkyl)amino,optionally substituted C₁₋₆-alkoxy, optionally substituted C₁₋₆-alkyl,optionally substituted C₂₋₆alkenyl, optionally substitutedC₂₋₆-alkenyloxy, optionally substituted C₂₋₆-alkynyl, and optionallysubstituted C₂₋₆-alkynyloxy, R³ is selected from hydrogen, optionallysubstituted C₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl, andoptionally substituted C₂₋₆-alkynyl, and R^(1*), R², R⁵, and R^(5*) eachdesignate hydrogen.
 91. A nucleoside analogue according to claim 83,wherein B designates a nucleobase, X is —O—, R² and R³ togetherdesignate a biradical selected from —(CH₂)₀₋₁—O—CH═CH—,—(CH₂)₀₋₁—S—CH═CH—, and —(CH₂)₀₋₁—N(R^(N))—CH═CH— where R^(N) isselected from hydrogen and C₁₋₄-alkyl, Q is selected from hydroxy,mercapto, C₁₋₆-alkylthio, amino, mono- or di(C₁₋₆-alkyl)amino,optionally substituted C₁₋₆-alkoxy, optionally substitutedC₂₋₆-alkenyloxy, optionally substituted C₂₋₆-alkynyloxy, monophosphate,diphosphate, and triphosphate, R³ is Q which is selected from hydrogen,azido, halogen, cyano, nitro, hydroxy, mercapto, C₁₋₆-alkylthio, amino,mono- or di(C₁₋₆-alkyl)amino, optionally substituted C₁₋₆-alkoxy,optionally substituted C₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl,optionally substituted C₂₋₆-alkenyloxy, optionally substitutedC₂₋₆-alkynyl, and optionally substituted C₂₋₆-alkynyloxy, and R^(1*),R^(2*), R^(4*), R⁵, and R^(5*) each designate hydrogen.
 92. A nucleosideanalogue according to claim 60, which is selected from(1R,3R,4R,7S)-7-(2-cyanoethoxy(diisopropylamino)phosphinoxy)-1-(4,4′-dimethoxytrityloxymethyl)-3-(thymin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane,(1R,3R,4R,7S)-7-hydroxy-1-(4,4′-dimethoxytrityloxymethyl)-3-(thymin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane-7-O-(2-chlorophenylphosphate), and(1R,3R,4R,7S)-7-hydroxy-1-(4,4′-dimethoxytrityloxymethyl)-3-(thymin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane-7-O—(H-phosphonate)and the 3-(cytosin-1-yl), 3-(urasil-1-yl), 3-(adenin-1-yl) and3-(guanin-1-yl) analogues thereof.
 93. A method of using an LNA asdefined in claim 60 for the preparation of an LNA modifiedoligonucleotide (an oligomer) as defined in claim
 1. 94. A methodaccording to claim 93, wherein the LNA modified oligonucleotidecomprises normal nucleosides as well as modified nucleosides differentfrom those defined in claim
 60. 95. A method according to claim 93,wherein the incorporation of LNA modulates the ability of theoligonucleotide to act as a substrate for nucleic acid active enzymes.96. A method of using of an LNA as defined in claim 60 for thepreparation of a conjugate of an LNA modified oligonucleotide and acompound selected from proteins, amplicons, enzymes, polysaccharides,antibodies, haptens, peptides, and PNA.
 97. A conjugate of an LNAmodified oligonucleotide (an oligomer) as defined in claim 1 and acompound selected from proteins, amplicons, enzymes, polysaccharides,antibodies, haptens, peptides, and PNA.
 98. A method of using an LNA asdefined in claim 60 as a substrate for enzymes active on nucleic acids.99. A method according to claim 98, wherein the substituent Q in theformula I in claim 60 designates a triphosphate,
 100. A method accordingto claim 98, wherein the LNA is used as a substrate for DNA and RNApolymerases.
 101. A method of using an LNA as defined in claim 60 as atherapeutic agent.
 102. A method of using an LNA as defined in claim 60for diagnostic purposes.
 103. A solid support material havingimmobilised thereto an optionally nucleobase protected and optionally5′-OH protected LNA.
 104. A method of using one or more LNA as definedin claim 60 in the construction of solid surface onto which LNA modifiedoligonucleotides of different sequences are attached.
 105. A methodaccording to claim 113, wherein the LNA modified oligonucleotides areattached in a predetermined pattern.
 106. A method according to claim113, wherein the LNAs are used to equalise the T_(m) of thecorresponding unmodified reference oligonucleotides.
 107. A methodaccording to claim 113, wherein the LNA modified oligonucleotides havean increased affinity toward complementary ssDNA or ssRNA compared tonative oligonucleotide.
 108. A method according to claim 113, whereinthe LNA modified oligonucleotides have an increased specificity towardcomplementary ssDNA or ssRNA compared to native oligonucleotide.
 109. Amethod of using LNA modified oligomers (ribozymes) as defined in claim 1in the sequence specific cleavage of target nucleic acids.
 110. A methodof using an LNA modified oligonucleotide (an oligomer) as defined inclaim 1 in therapy.
 111. A method according to claim 110, wherein theLNA modified oligonucleotide recruits RNAseH.
 112. A method of usingcomplexes of more than one LNA modified oligonucleotide (an oligomer) asdefined in claim 1 in therapy.
 113. A method of using an LNA modifiedoligonucleotide (an oligomer) as defined in claim 1 as an aptamer intherapeutic applications.
 114. A method according to claim 119, whereinthe LNA modified oligonucleotide comprises at least one internucleosidelinkage not being a phosphate diester linkage.
 115. A method of using anLNA modified oligonucleotide (an oligomer) as defined in claim 1 indiagnostics.
 116. A method according to claim 115, wherein theoligonucleotide comprises a photochemically active group, athermochemically active group, a chelating group, a reporter group, or aligand that facilitates the direct of indirect detection of theoligonucleotide or the immobilisation of the oligonucleotide onto asolid support.
 117. A method according to claim 116, wherein thephotochemically active group, the thermochemically active group, thechelating group, the reporter group, or the ligand includes a spacer(K), said spacer comprising a chemically cleavable group.
 118. A methodaccording to claim 116, wherein the photochemically active group, thethermochemically active group, the chelating group, the reporter group,or the ligand is attached via the biradical (i.e. as R*) of at least oneof the LNA(s) of the oligonucleotide.
 119. A method according to claim115 for capture and detection of naturally occurring or synthetic doublestranded or single stranded nucleic acids.
 120. A method according toclaim 115 for purification of naturally occurring double stranded orsingle stranded nucleic acids.
 121. A method according to claim 115 as aprobe in in-situ hybridisation, in Southern hydridisation, Dot blothybridisation, reverse Dot blot hybridisation, or in Northernhybridisation.
 122. A method according to claim 115 in the constructionof an affinity pair.
 123. A method according to claim 115 as a primer ina nucleic acid sequencing reaction or primer extension reactions.
 124. Amethod according to claim 115 as a primer in a nucleic acidamplification reaction.
 125. A method according to claim 124, whereinthe primer is so adapted that the amplification reaction is anessentially linear reaction.
 126. A method according to claim 124,wherein the primer is so adapted that the amplification reaction is anessentially exponential reaction.
 127. A method according to claim 124,wherein the nucleic acid amplification reaction results in a doublestranded DNA product comprising at least one single stranded end.
 128. Amethod of using an LNA modified oligonucleotide (an oligomer) as definedin claim 1 as an aptamer in molecular diagnostics.
 129. A method ofusing an LNA modified oligonucleotide (an oligomer) as defined in claim1 as an aptamer in RNA mediated catalytic processes.
 130. A method ofusing an LNA modified oligonucleotide (an oligomer) as defined in claim1 as an aptamer in specific binding of antibiotics, drugs, amino acids,peptides, structural proteins, protein receptors, protein enzymes,saccharides, polysaccharides, biological cofactors, nucleic acids, ortriphosphates.
 131. A method of using an LNA modified oligonucleotide(an oligomer) as defined in claim 1 as an aptamer in the separation ofenantiomers from racemic mixtures by stereospecific binding.
 132. Amethod of using an LNA modified oligonucleotide (an oligomer) as definedin claim 1 for the labelling of cells.
 133. A method according to claim132, wherein the label allows the cells to be separated from unlabelledcells.
 134. A method of using an LNA modified oligonucleotide (anoligomer) as defined in claim 1 to hybridise to non-protein codingcellular RNAs in vivo or in-vitro.
 135. A method of using an LNAmodified oligonucleotide (an oligomer) as defined in claim 1 in theconstruction of an oligonucleotide containing a fluorophor and aquencher, positioned in such a way that the hybridised state of theoligonucleotide can be distinguished from the unbound state of theoligonucleotide by an increase in the fluorescent signal from the probe.136. A method of using an LNA modified oligonucleotide (an oligomer) asdefined in claim 1 in the construction of Taqman probes or MolecularBeacons.
 137. A kit for the isolation, purification, amplification,detection, identification, quantification, or capture of natural orsynthetic nucleic acids, the kit comprising a reaction body and one ormore LNA modified oligonucleotides (oligomer) as defined in claim 1.138. A kit according to claim 137, wherein the LNA modifiedoligonucleotides are immobilised onto said reactions body.
 139. A kitfor the isolation, purification, amplification, detection,identification, quantification, or capture of natural or syntheticnucleic acids, the kit comprising a reaction body and one or more LNAsas defined in claim
 60. 140. A kit according to claim 139, wherein theLNAs are immobilised onto said reactions body.